Patent Application
20200255846
The invention relates to methods for increasing plant yield, and in particular seed yield by reducing the expression of GSE5 or GSE5-Like in a plant. Also described are genetically altered plants characterised by the above phenotype and methods of producing such plants.
Modern agriculture must meet the challenges of feeding an increasing population and decreasing arable land. Rice is an important crop, providing food for more than half the global population. The genetic variation in diverse rice varieties provides a valuable resource to improve important agronomic traits in rice. Rice breeders have explored natural variation in genes involved in the regulation of yield-related traits to develop elite rice varieties (Zuo and Li, 2014). Rice grain yield is determined by grain weight, grain number per panicle and panicle number per plant. Grain size is associated with grain weight, grain yield and appearance quality. Several QTL genes for grain size have been identified in rice (Che et al., 2015; Duan et al., 2015; Fan et al., 2006; Hu et al., 2015; Ishimaru et al., 2013; Li et al., 2011; Qi et al., 2012; Shomura et al., 2008; Si et al., 2016; Song et al., 2007; Wang et al., 2015a; Wang et al., 2012; Wang et al., 2015b; Weng et al., 2008; Zhang et al., 2012), but only a few of these beneficial alleles are widely utilized by rice breeders (Li and Li, 2016; Zuo and Li, 2014).
Asian cultivated rice includes indica and japonica subspecies, which show large variation in grain size and shape. Typical indica varieties produce long grains, whereas japonica varieties form round and short grains. Natural variation in several genes has been reported to be selected by rice breeders. For example, natural variation in the major QTL for grain length (GS3) contributes to grain-length differences between indica varieties and japonica varieties (Fan et al., 2006; Mao et al., 2010). The indica varieties with long grains usually contain a loss-of-function allele, while japonica varieties with short grains often have the wild-type allele. By contrast, the major QTL gene for grain width (qSW5/GW5) influences grain-width differences between indica varieties and japonica varieties. The qSW5/GW5 encodes an unknown protein (Shomura et al., 2008; Weng et al., 2008). The 1212-bp deletion in most japonica varieties disrupts the qSW5 gene, resulting in wide grains. By contrast, some indica varieties do not contain this 1212-bp deletion in the qSW5 gene, thereby producing narrow grains (Weng et al., 2008). Genome-wide association studies (GWAS) have identified multiple association signals for grain size in cultivated rice (Huang et al., 2010). The QTL gene GLW7/OsSPL13 has been recently identified using the GWAS approach (Si et al., 2016). High expression of GLW7 is associated with large grains in tropical japonica rice. However, the grain size genes underlying natural variation have not been fully explored in rice.
Here we identify a novel quantitative trait locus for grain size (GSE5) using a genome-wide association study with functional testing. GSE5 encodes a plasma membrane associated protein with IQ domains (IQD), which regulates grain width by restricting cell proliferation. Loss-of-function of GSE5 increases grain width, while overexpression of GSE5 results in slender grains. Two major type deletions (DEL1 and DEL2) happen in the promoter region of GSE5 in some indica varieties and most japonica varieties, respectively, resulting in the decreased expression of GSE5 and wide grains. DEL1 and DEL2 are widely utilized in indica and japonica rice production, respectively. Wild rice accessions contain DEL1 and DEL2, suggesting that these two deletions in cultivated rice are likely to have originated from different wild rice accessions during rice domestication. We have also identified a GSE5-Like protein, that has 72.5% identity with GSE5 and that similarly, reducing the expression of GSE5-Like increases grain length, grain width and yield. Thus, our findings provide insight into a natural variation in grain size control.
As seed yield is a major factor in determining the commercial success of grain crops it is important to not only understand the genetic factors that underlie this trait, but also how to modulate such factors to improve overall grain yield. The present invention addresses this need.
The inventors have surprisingly identified that the expression of GSE5 or GSE5-Like correlates negatively with the yield component traits, grain weight, grain width and thousand kernel weight (TKW) across Oryza sativa accessions. Accordingly, the inventors have surprisingly shown that reducing the level of GSE5 or GSE5-Like expression and/or the activity of the GSE5 or GSE5-Like polypeptide can significantly increase grain yield.
In one aspect of the invention there is provided a method of increasing yield in a plant, the method comprising reducing or abolishing the expression of at least one (grain size on chromosome 5) GSE5 or GSE5-Like nucleic acid and/or reducing the activity of a GSE5 or GSE5-Like polypeptide in said plant. In one embodiment, the method may comprise reducing or abolishing the expression of at least one GSE5 and GSE5-Like nucleic acid and/or reducing the activity of a GSE5 and GSE5-Like polypeptide in said plant.
In one embodiment, said increase is an increase in grain yield. Preferably, said increase in grain yield is preferably an increase in at least one of grain weight, grain width and/or thousand kernel weight.
In one embodiment, the method comprises introducing at least one mutation into the nucleic acid sequence encoding GSE5 or GSE5-Like or at least one mutation into the promoter of GSE5 or GSE5-Like. Preferably, said mutation is a loss of function or partial loss of function mutation. More preferably, said mutation is an insertion, deletion and/or substitution.
In one embodiment, the GSE5 nucleic acid encodes a polypeptide comprising SEQ ID NO: 1 or a functional variant or homolog thereof. Preferably, the GSE5 nucleic acid comprises SEQ ID NO: 2 or a functional variant or homolog thereof. In another embodiment, the GSE5-Like nucleic acid encodes a polypeptide comprising SEQ ID NO: 57 or a functional variant or homolog thereof. Preferably, the GSE5-Like nucleic acid comprises SEQ ID NO: 55 or 56 or a functional variant or homolog thereof.
In another embodiment, the GSE5 promoter comprises a nucleic acid sequence as defined in SEQ ID NO: 28 or a functional variant or homolog thereof.
In one embodiment, the mutation is introduced using targeted genome modification, preferably ZFNs, TALENs or CRISPR/Cas9. In an alternative embodiment, the mutation is introduced using mutagenesis, preferably TILLING or T-DNA insertion. In a further alternative embodiment, the method comprises using RNA interference to reduce or abolish the expression of a GSE5 nucleic acid and/or reduce or abolish the activity of a GSE5 or GSE5-Like promoter.
In one embodiment, said increase in seed yield is relative to a control or wild-type plant.
In another aspect of the invention, there is provided a genetically modified plant, plant cell or part thereof characterised by a reduced level of GSE5 or GSE5-Like nucleic acid expression and/or reduced activity of the GSE5 or GSE5-Like polypeptide.
In one embodiment, said plant is characterised by an increase in yield compared to a wild-type on control pant. Preferably, said increase in yield is an increase in at least grain yield. More preferably, said increase in grain yield is preferably an increase in at least one of grain weight, grain width and/or thousand kernel weight.
In one embodiment, said plant comprises at least one mutation in at least one nucleic acid sequence encoding GSE5 or GSE5-Like or at least one mutation in the promoter of GSE5 or GSE5-Like. Preferably, said mutation is a loss of function or partial loss of function mutation. More preferably, said mutation is an insertion, deletion and/or substitution.
In one embodiment, the GSE5 nucleic acid encodes a polypeptide comprising of SEQ ID NO: 1 or a functional variant or homolog thereof. Preferably, the GSE5 nucleic acid comprises SEQ ID NO: 2 or 32 or a functional variant or homolog thereof. In another embodiment, the GSE5-Like nucleic acid encodes a polypeptide comprising SEQ ID NO: 57 or a functional variant or homolog thereof. Preferably, the GSE5-Like nucleic acid comprises SEQ ID NO: 55 or 56 or a functional variant or homolog thereof.
In another embodiment, the GSE5 promoter comprises a nucleic acid sequence as defined in SEQ ID NO: 28 or a functional variant or homolog thereof.
In one embodiment, the mutation is introduced using targeted genome modification, preferably ZFNs, TALENs or CRISPR/Cas9. In another embodiment, the mutation is introduced using mutagenesis, preferably TILLING or T-DNA insertion. In a further alternative embodiment, the plant comprises an RNA interference construct that reduces or abolishes the expression of a GSE5 or GSE5-Like nucleic acid and/or reduces or abolishes the activity of a GSE5 or GSE5-Like promoter.
In one embodiment, the plant part is a seed.
In another aspect of the invention, there is provided a method of producing a plant with increased yield, the method comprising introducing at least one mutation into at least one nucleic acid sequence encoding GSE5 or GSE5-Like and/or at least one mutation in the promoter of GSE5 or GSE5-Like. Preferably, the mutation is a loss of function or partial loss of function mutation. More preferably, the mutation is an insertion, deletion and/or substitution.
In one embodiment, the mutation is introduced using mutagenesis or targeted genome modification. Preferably, the targeted genome modification is selected from ZFNs, TALENs or CRISPR/Cas9.
In one embodiment, mutagenesis is selected from TILLING or T-DNA insertion.
In another aspect of the invention, there is provided a plant, plant part or plant cell obtained by the method described herein. In a further aspect of the invention, there is provided a seed obtained or obtainable from the plant as described herein or the method as described herein.
In a further aspect of the invention, there is provided a method for identifying and/or selecting a plant that will have an increased seed yield phenotype, the method comprising detecting in the plant or plant germplasm at least one mutation in the promoter of the GSE5 or GSE5-Like gene, wherein said plant or progeny thereof is selected.
In one embodiment, the mutation is an insertion and/or deletion. Preferably, the mutation is the deletion of a nucleic acid sequence comprising SEQ ID NO: 29 (DEL1) or SEQ ID NO: 30 (DEL2). Alternatively or additionally, the mutation is the insertion of a nucleic acid sequence comprising SEQ ID NO: 31 (IN1).
In a further embodiment, the method further comprises introgressing the chromosomal region comprising at least one of said polymorphisms and/or deletions into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.
In another aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding at least one DNA-binding domain that can bind to at least one GSE5 gene or GSE5-Like, wherein said sequence is selected from SEQ ID NOs: 15 to 20, 48, 51, 76 and 79 to 84.
In one embodiment, the nucleic acid sequence encodes at least one protospacer element, and wherein the sequence of the protospacer element is selected from SEQ ID NOs: 21 to 26 or 52 or 77 or a sequence that is at least 90% identical to SEQ ID NOs: 21 to 26 or 52 or 77.
In a further embodiment, the construct further comprises a nucleic acid sequence encoding a CRISPR RNA (crRNA) sequence, wherein said crRNA sequence comprises the protospacer element sequence and additional nucleotides.
In another embodiment, the construct further comprises a nucleic acid sequence encoding a transactivating RNA (tracrRNA).
In a further embodiment, the construct encodes at least one single-guide RNA (sgRNA), wherein said sgRNA comprises the tracrRNA sequence and the crRNA sequence, wherein the sgRNA.
Preferably the nucleic acid encoding a DNA-binding domain, protospacer element, crRNA, tracrRNA or sgRNA is operably linked to a promoter. Preferably, the promoter is a constitutive promoter.
In a further embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding a CRISPR enzyme. Preferably, the CRISPR enzyme is a Cas protein. More preferably, the Cas protein is Cas9 or a functional variant thereof.
In an alternative embodiment, the nucleic acid construct encodes a TAL effector. Preferably, the nucleic acid construct further comprises a sequence encoding an endonuclease or DNA-cleavage domain thereof. More preferably, the endonuclease is Fokl.
In another aspect of the invention there is provided a single guide (sg) RNA molecule wherein said sgRNA comprises a crRNA sequence and a tracrRNA sequence, wherein the crRNA sequence can bind to at least one sequence selected from SEQ ID NOs: 15 to 20, 48, 51, 76 or 79 to 84.
In a further aspect of the invention there is provided an isolated plant cell transfected with at least one nucleic acid construct as described herein.
In an alternative aspect of the invention there is provided an isolated plant cell transfected with at least a first nucleic acid construct as described herein (comprising nucleic acid encoding a sgRNA) and a second nucleic acid construct, wherein said second nucleic acid construct comprising a nucleic acid sequence encoding a Cas protein, preferably a Cas9 protein or a functional variant thereof. Preferably, the second nucleic acid construct is transfected before, after or concurrently with the first nucleic acid construct.
In another aspect of the invention, there is provided a genetically modified plant, wherein said plant comprises the transfected cell above. In one embodiment, the nucleic acid encoding the sgRNA and/or the nucleic acid encoding a Cas protein is integrated in a stable form.
In a further aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide as defined in SEQ ID NO: 1 or a functional variant or homolog thereof, wherein said sequence is operably linked to a regulatory sequence, wherein preferably said regulatory sequence is a tissue-specific promoter.
In another aspect of the invention there is provided a vector comprising the nucleic acid construct as described herein. In a further aspect, there is provided a host cell comprising the nucleic acid construct as described herein. In a yet further aspect, there is provided a transgenic plant expressing the nucleic acid construct as described herein.
In another aspect of the invention, there is provided a method of increasing grain length, the method comprising introducing and expressing in said plant the nucleic acid construct as described herein, wherein said increase is relative to a control or wild-type plant.
In a further aspect, there is provided a method for producing a plant with increased grain length, the method comprising introducing and expressing in said plant the nucleic acid construct as described herein, wherein said increase is relative to a control or wild-type plant.
In another aspect, there is provided a plant obtained or obtainable by the method as described herein.
In another aspect of the invention, there is provided the use of a nucleic acid construct as described herein to modulate the expression levels of at least one GSE5 or GSE5-Like nucleic acid in a plant. Preferably said nucleic acid construct reduces the expression levels of at least one GSE5 or GSE5-Like nucleic acid in a plant. Alternatively, said nucleic acid construct increases the expression levels of at least one GSE5 or GSE5-Like nucleic acid in a plant.
In a final aspect of the invention, there is provided a method for obtaining the genetically modified plant as described above, the method comprising:
In one embodiment of any of the above aspects, the plant is a crop plant. Preferably, the crop plant is selected from rice, wheat, maize, soybean and sorghum. More preferably, the crop plant is rice, preferably the japonica or indica variety.
The invention is further described in the following non-limiting figures:
(a) Genome-wide association study of grain width. Manhattan plots for grain width. Dashed line represents the significance threshold (P=2.78×10−5). The arrows indicate the loci for grain width.
(b) Q local manhattan plot (top) and LD heatmap (bottom) surrounding the peak on Chromosome 5. Dashed lines indicate the candidate region for the peak.
(c) The schematic diagram of the 22.42-kb genomic region. This region contains qSW5 and LOC_Os05g09520. Most japonica varieties have a 1212-bp deletion (DEL2) in the qSW5 gene. Some indica varieties have no deletion in qSW5, while some indica varieties contain a 950-bp deletion (DEL1) in the 3′ flanking region of qSW5, a 367-bp insertion (IN1) in the 5′ flanking region of LOC_Os05g09520 and a nucleotide change (G/A) in the first exon of LOC_Os05g09520. The arrow shows the direction of the qSW5 transcription. The red dash lines represent the deletions in the genomic regions.
(d) Comparison of qSW5 expression in young panicles of indica varieties without (1) or with (2) the 950-bp deletion (DEL1) in the 3′ flanking region of qSW5 (n=34/36).
(e) Correlation of the 950-bp deletion (DEL1) and 367-bp insertion (IN1) with grain width. Mature grains from the indica varieties without (1) or with DEL1+IN1 (2) were measured (n=68/65).
Values (d and e) are means±SD. Significance is determined using analysis of variance (ANOVA) (**P<0.01).
(a) Comparison of LOC_Os05g09520 expression in young panicles of narrow grain (NGV) and wide grain (WGV) indica varieties. Values are means±SD (n=20/20). Significance is determined using analysis of variance (ANOVA) (*P<0.05).
(b) Comparison of LOC_Os05g09520 expression in young panicles of rice varieties without (1) or with DEL1+IN1 (2) and DEL2 (3). Values are means±SD (n=34/36/31). Significance is determined using analysis of variance (ANOVA) (*P<0.05).
(c) Expression levels of LOC_Os05g09520 expression in young panicles of the japonica variety Nipponbare (NIP) with DEL2 and its near isogenic line (NIL). NIL contains the LOC_Os05g09520 allele from the narrow grain indica variety 93-11 in the japonica variety Nipponbare background. Values are means±SE (n=3). Significance is determined using t-test (**P<0.01).
(d) The constructs for each of the promoter-luciferase (LUC) fusions are shown. The arrow shows the direction of the qSW5 transcription.
(e) Effects of DEL1, IN1 and DEL2 on the activity of the GSE5 promoter. N. benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harboring proGSE5:LUC (1), proGSE5DEL1+IN1:LUC (2), proGSE5DEL1:LUC (3) and proGSE5DEL2:LUC (4) plasmids, respectively. Relative reporter activity (LUC/REN) was calculated, and the value for proGSE5:LUC sets at 100. Values are means±SE (n=3). Significance is determined using t-test (**P<0.01).
(a) The GSE5-cr mutant was generated by CRISPR/Cas9. In GSE5-cr mutant, the 1-bp deletion happens in the first exon of GSE5, resulting in a reading frame shift.
(b) Grains of Zhonghua 11 (ZH11) (left) and GSE5-cr (right).
(c-e) Grain width (c), grain length (d) and thousand grain weight (e) of Zhonghua 11 (ZH11) and GSE5-cr.
(f) Grains of Zhonghua 11 (ZH11) (left) and proActin:GSE5 (right). GSE5 was overexpressed in ZH11 background.
(g, h) Grain width (g) and grain length (h) of Zhonghua 11 (ZH11) and proActin:GSE5. GSE5 was overexpressed in ZH11 background.
(i, j) Grain width (i) and grain length (j) of Nipponbare (NP) and a near isogenic line (NIL), which contains the GSE5 locus from the narrow grain indica variety 93-11 in the japonica variety Nipponbare background.
Values (c-e, g-j) are means±SE. Significance is determined using t-test (**P<0.01). Bars=1 mm in b, f.
(a, b) The outer epidermal surface of ZH11 (a) and GSE5-cr (b).
(c, d) The outer epidermal cell width (c) and the calculated outer epidermal cell number
(d) of ZH11 and GSE5-cr lemma in the grain-width direction.
(e, f) The outer epidermal cell width (e) and the calculated outer epidermal cell number
(f) of ZH11 and proActin:GSE5 (OE) lemma in the grain-width direction.
(g, h) The outer epidermal cell length (g) and the calculated outer epidermal cell number (h) of ZH11 and proActin:GSE5 (OE) lemma in the grain-length direction.
Values (c-h) are means±SE. Significance is determined using t-test (**P<0.01). Bars=100 μm in a and b.
(a) The GSE5 protein contains two IQ motifs and an unknown DUF4005 domain.
(b) The bimolecular fluorescence complementation (BiFC) assays show that GSE5 associated with OsCaM1-1 in N. benthamiana. nYFP-OsCaM1-1 and cYFP-GSE5 were coexpressed in leaves of N. benthamiana.
(c) Quantitative real-time RT-PCR analysis of GSE5 expression in young panicles of 5 cm (YP5), 10 cm (YP10), 15 cm (YP15) and 20 cm (YP20). Values are given as mean±SE (n=3).
(d-h) GSE5 expression activity was monitored using proGSE5:GSE5-GUS transgenic plants. GUS activity was detected in developing panicles.
(i) Subcellular localization of GSE5-GFP in proGSE5:GSE5-GFP transgenic plants. GFP fluorescence in proGSE5:GSE5-GFP transgenic plants was detected in the cell periphery. FM4-64 was used to stain the membrane.
(j) Cells were plasmolysed with 30% sucrose. GSE5-GFP was detected in the shrunken plasma membrane. FM4-64 was used to stain the membrane.
Bars=50 μm in b, 1 mm in d and e, 1 cm in f and g, 5 cm in h, and 10 μm in i and j.
(c) Geographical origin of wild rice accessions used in this study. Wild rice accessions (O. rufipogon) contained GSE5, GSE5DEL1+IN1 and GSE5DEL2 haplotypes.
(d) Phylogenetic tree. The approximate 8.4 kb sequences including 6320-bp 5′ flanking sequence, the GSE5 gene and 1580-bp 3′ flanking sequence from 63 cultivated rice with GSE5, GSE5DEL1+IN1 and GSE5DEL2 haplotypes and 26 O. rufipogon with GSE5, GSE5DEL1+IN1 and GSE5DEL2 haplotypes were used to construct phylogenetic tree. Bootstrap values over 60% are given on the branches. The red letters represent O. rufipogon accession.
Values are given as mean±SE. **P<0.01 compared with parental line (ZH11) by Student's t-test.
The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature.
As used herein, the words “nucleic acid”, “nucleic acid sequence”, “nucleotide”, “nucleic acid molecule” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term “gene” or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.
The terms “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
The aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.
Accordingly, in a first aspect of the invention, there is provided a method of increasing yield in a plant, the method comprising reducing or abolishing the expression of at least one nucleic acid encoding a grain size on chromosome 5 (referred to herein as GSE5) or GSE5-Like polypeptide and/or reducing the activity of a GSE5 polypeptide or GSE5-Like polypeptide in said plant. In one embodiment, the method may comprise reducing or abolishing the expression of at least one GSE5 and GSE5-Like nucleic acid and/or reducing the activity of a GSE5 and GSE5-Like polypeptide in said plant.
The term “yield” in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight. Alternatively, the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters. Preferably, in the present context, the term “yield” of a plant relates to propagule generation (such as seeds) of that plant. Thus, in a preferred embodiment, the method relates to an increase in seed yield or total seed yield.
The terms “seed” and “grain” as used herein can be used interchangeably.
According to the invention, seed yield can be measured by assessing one or more of seed weight, seed size, seed number per pod, seed number per plant, pod length, seed protein, a combination of both seed size and seed number and/or lipid content and weight of seed per pod. However, seed width and weight are some of the main components that contribute to seed yield. Therefore, in one embodiment an increase in seed yield comprises an increase in seed biomass or seed weight, which may be an increase in the seed weight per plant or in an increase in individual seed weight, an increase in seed width (individual or as an average over the whole plant) and/or an increase in thousand kernel weight (TKW), which can be extrapolated from the number of filled seeds counted and their total weight. An increase in the TKW can result from an increase in seed size and/or seed weight. Preferably, an increase in seed yield is an increase in at least one of seed weight, seed width and TKW. Yield is increased relative to control plants. The skilled person would be able to measure any of the above seed yield parameters using known techniques in the art.
The terms “increase”, “improve” or “enhance” as used herein are interchangeable. In one embodiment, seed yield, and preferably seed weight, seed width and/or the TKW are increased by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 120% or more in comparison to a control plant. Preferably, the increase is at least 2-10%, more preferably 3-8%. These increases can be measured by any standard technique known to the skilled person. In one embodiment, seed width is increased by more than 100%, preferably at least 110% or more compared to a control phenotype.
The terms “reducing” means a decrease in the levels of GSE5 or GSE5-Like polypeptide expression and/or activity by up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a wild-type or control plant. In a preferred embodiment, said decrease is at least 30%. The term “abolish” expression means that no expression of GSE5 or GSE5-Like polypeptide is detectable or that no functional GSE5 or GSE5-Like polypeptide is produced. Method for determining the level of GSE5 or GSE5-Like polypeptide expression and/or activity would be well known to the skilled person. These reductions can be measured by any standard technique known to the skilled person. For example, a reduction in the expression and/or content levels of at least GSE5 or GSE5-Like expression may be a measure of protein and/or nucleic acid levels and can be measured by any technique known to the skilled person, such as, but not limited to, any form of gel electrophoresis or chromatography (e.g. HPLC).
By “at least one mutation” is means that where the GSE5 or GSE5-Like gene is present as more than one copy or homologue (with the same or slightly different sequence) there is at least one mutation in at least one gene. Preferably all genes are mutated.
Grain size and weight are important agronomic traits in crops. We have identified a novel grain size gene (GSE5) that encodes a plasma membrane-associated protein with IQ domains (IQD), which interacts with calmodulin (OsCaM1-1). In rice, loss of GSE5 function causes wide and heavy grains, while overexpression of GSE5 results in narrow and long grains. We have also identified a GSE5-Like protein, that has 72.5% identity with GSE5 and that similarly, a loss of GSE5-Like function increases grain length, grain width and yield. By performing a BLAST search in the databases, we found that GSE5 and GSE5-Like shares significant similarity with its homologs in other crops, such as maize, wheat, sorghum and brachypodium. Our current knowledge of GSE5 and GSE5-Like function suggests that GSE5 and GSE5-Like and its homologs in other crops or plant species can be used to engineer large and heavy seeds in these key crops. We can also use CRISPR/Cas9 technology to knock-out GSE5 or GSE5-Like or its homologs in other crops to increase seed size and weight in these crops. We also can also use RNAi technology to knock-down the expression of GSE5 or GSE5-Like or its homologs in crops to increase seed size and weight in these crops.
In one embodiment, the method comprises introducing at least one mutation into the, preferably endogenous, gene encoding GSE5 or GSE5-Like and/or the GSE5 or GSE5-Like promoter. Preferably said mutation is in the coding region of the GSE5 or GSE5-Like gene. In a further embodiment, at least one mutation or structural alteration may be introduced into the GSE5 or GSE5-Like promoter such that the GSE5 or GSE5-Like gene is either not expressed (i.e. expression is abolished) or expression is reduced, as defined herein. In an alternative embodiment, at least one mutation may be introduced into the GSE5 or GSE5-Like gene such that the altered gene does not express a full-length (i.e. expresses a truncated) GSE5 or GSE5-Like protein or does not express a fully functional GSE5 or GSE5-Like protein. In this manner, the activity of the GSE5 or GSE5-Like polypeptide can be considered to be reduced or abolished as described herein. In any case, the mutation may result in the expression of GSE5 or GSE5-Like with no, significantly reduced or altered biological activity in vivo. Alternatively, GSE5 or GSE5-Like may not be expressed at all.
In another embodiment, the sequence of the GSE5 gene comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 2 (cDNA) or 32 (genomic) or a functional variant or homologue thereof and encodes a polypeptide as defined in SEQ ID NO: 1 or a functional variant or homologue thereof.
In another embodiment, the sequence of the GSE5-Like gene comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 55 (cDNA) or 56 (genomic) or a functional variant or homologue thereof and encodes a polypeptide as defined in SEQ ID NO: 57 or a functional variant or homologue thereof.
By “GSE5 promoter” is meant a region extending for at least 6320 bp upstream of the ATG codon of the GSE5 ORF (open reading frame). In one embodiment, the sequence of the GSE5 promoter comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 28 or a functional variant or homologue thereof. By “GSE5-Like” promoter is meant a region extending at least 2 kb, preferably 6 kb upstream of the GSE5-Like ORF.
In the above embodiments an ‘endogenous’ nucleic acid may refer to the native or natural sequence in the plant genome. In one embodiment, the endogenous sequence of the GSE5 gene comprises SEQ ID NOs: 2 or 32 and encodes an amino acid sequence as defined in SEQ ID NO: 1 or homologs thereof. Similarly, the endogenous sequence of the GSE5-Like gene comprises SEQ ID NOs: 55 or 56 and encodes an amino acid sequence as defined in SEQ ID NO: 57 or homologs thereof. Also included in the scope of this invention are functional variants (as defined herein) and homologs of the above identified sequences. Examples of GSE5 homologs are shown in SEQ ID NOs: 3 to 10. Accordingly, in one embodiment, the homolog encodes a polypeptide selected from SEQ ID NOs: 3, 5, 7 and 9 or the homolog comprises or consists of a nucleic acid sequence selected from SEQ ID NOs: 4, 6, 8 and 10. Examples of GSE5-Like homologs are shown in SEQ ID NOs: 58 to 75. Accordingly, in one embodiment, the homolog encodes a polypeptide selected from SEQ ID NOs: 60, 63, 66, 69, 72 and 75 or the homolog comprises or consists of a nucleic acid sequence selected from SEQ ID NOs: 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73 and 74.
The term “functional variant of a nucleic acid sequence” as used herein with reference to any of SEQ ID NOs: 1 to 88 refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence. A functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
In one embodiment, a functional variant has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid or amino acid sequence.
The term homolog, as used herein, also designates a GSE5 or GSE5-Like promoter or GSE5 or GSE5-Like gene orthologue from other plant species. A homolog may have, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the amino acid represented by any of SEQ ID NO: 1 or 57 or to the nucleic acid sequences as shown by SEQ ID NOs: 2, 32, 55 or 56. In one embodiment, overall sequence identity is at least 37%. In one embodiment, overall sequence identity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
Functional variants of GSE5 or GSE5-Like homologs as defined above are also within the scope of the invention.
The “GSE5” or “grain size on chromosome 5” gene encodes a plasma membrane associated protein. This protein is characterised by a IQ calmodulin-binding motif or IQD.
Accordingly, in one embodiment, the GSE5 nucleic acid (coding) sequence encodes a GSE5 protein comprising a IQD domain as defined below, or a variant thereof, wherein the variant has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the IQD as defined herein. In a preferred embodiment, the GSE5 polypeptide is characterised by at least one IQD with at least 75% homology thereto.
In one embodiment, the sequence of the IQD is as follows:
Wherein x is any amino acid.
Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.
Suitable homologues can be identified by sequence comparisons and identifications of conserved domains. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example when overexpressed in a plant.
Thus, the nucleotide sequences of the invention and described herein can also be used to isolate corresponding sequences from other organisms, particularly other plants, for example crop plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Topology of the sequences and the characteristic domains structure can also be considered when identifying and isolating homologs. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen plant. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
In a further embodiment, a variant as used herein can comprise a nucleic acid sequence encoding a GSE5 or GSE5-Like polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to a nucleic acid sequence as defined in SEQ ID NO: 2 or 32 or 55 or 56.
In one embodiment, there is provided a method of increasing yield in a plant, as described herein, the method comprising reducing or abolishing the expression of at least one nucleic acid encoding a GSE5 or GSE5-Like polypeptide, as described herein, wherein the method comprises introducing at least one mutation into at least
GSE5 or GSE5-Like gene and/or promoter, wherein the GSE5 or GSE5-Like gene comprises or consists of
In a preferred embodiment, the mutation that is introduced into the endogenous GSE5 or GSE5-Like gene or promoter thereof to silence, reduce, or inhibit the biological activity and/or expression levels of the GSE5 or GSE5-Like gene or protein can be selected from the following mutation types
As used herein, an “insertion” may refer to the insertion of at least one nucleotide. In one embodiment said insertion may be between 20 and 500 base pairs, more preferably between 300 and 400 base pairs.
As used herein, a “deletion” may refer to the deletion of at least one nucleotide. In one embodiment, said deletion may be between 1 and 1500 base pairs, more preferably between 900 and 1300 base pairs.
In general, the skilled person will understand that at least one mutation as defined above and which leads to the insertion, deletion or substitution of at least one nucleic acid or amino acid compared to the wild-type GSE5 promoter or GSE5 or GSE5-Like nucleic acid or protein sequence can affect the biological activity of the GSE5 or GSE5-Like protein.
In one embodiment, the mutation is introduced into the IQ domain of GSE5. Preferably, said mutation is a loss of function mutation such as a premature stop codon, or an amino acid change in a highly conserved region that is predicted to be important for protein structure.
In another embodiment, the mutation is introduced into the GSE5 or GSE5-Like promoter and is at least the deletion and/or insertion of at least one nucleic acid. In one embodiment, a sequence comprising or consisting of SEQ ID NO: 29 or 30 or a variant thereof is deleted. In a further or alternative embodiment a sequence comprising or consisting of SEQ ID NO: 31 or a variant thereof is inserted. Other major changes such as deletions that remove functional regions of the promoter are also included as these will reduce the expression of GSE5.
In one embodiment a mutation may be introduced into the GSE5 or GSE5-Like promoter and at least one mutation is introduced into the GSE5 or GSE5-Like gene.
In one embodiment, the mutation is introduced using mutagenesis or targeted genome editing. That is, in one embodiment, the invention relates to a method and plant that has been generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties.
Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. To achieve effective genome editing via introduction of site-specific DNA DSBs, four major classes of customisable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). Meganuclease, ZF, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.
Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem, 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats.
These repeats only differ from each other by two adjacent amino acids, their repeat-variable di-residue (RVD). The RVD that determines which single nucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide, with the four most common RVDs each preferentially associating with one of the four bases. Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity. TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nuclease (TALEN) which makes targeted DNA double-strand breaks (DSBs) in vivo for genome editing. The use of this technology in genome editing is well described in the art, for example in U.S. Pat. Nos. 8,440,431, 8,440,432 and 8,450,471. Cermak T et al. describes a set of customized plasmids that can be used with the Golden Gate cloning method to assemble multiple DNA fragments. As described therein, the Golden Gate method uses Type IIS restriction endonucleases, which cleave outside their recognition sites to create unique 4 bp overhangs. Cloning is expedited by digesting and ligating in the same reaction mixture because correct assembly eliminates the enzyme recognition site. Assembly of a custom TALEN or TAL effector construct and involves two steps: (i) assembly of repeat modules into intermediary arrays of 1-10 repeats and (ii) joining of the intermediary arrays into a backbone to make the final construct. Accordingly, using techniques known in the art it is possible to design a TAL effector that targets a GSE5 or GSE5-Like gene or promoter sequence as described herein.
Another genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in U.S. Pat. No. 8,697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (I-III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.
One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted (Wiles et al., 2015).
Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.
The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5′ end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3. Accordingly, using techniques known in the art it is possible to design sgRNA molecules that target a GSE5 or GSE5-Like gene or promoter sequence as described herein. In one embodiment, the sgRNA molecules target a sequence selected from SEQ ID No: 15 to 20, 48, 51, 76 or 79 to 84 or a variant thereof as defined herein. In a further embodiment, the sgRNA molecules comprises a protospacer sequence selected from SEQ ID NO: 21 to 26 and 52 and 77 or a variant thereof, as defined herein. In a further embodiment, the sgRNA nucleic acid sequence comprises a sequence comprising or consisting of SEQ ID NO: 78 or 89 or a variant thereof, as defined herein.
Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.
In one embodiment, the method uses the sgRNA constructs defined in detail below to introduce a targeted mutation into a GSE5 or GSE5-Like gene and/or promoter.
Alternatively, more conventional mutagenesis methods can be used to introduce at least one mutation into a GSE5 or GSE5-Like gene or GSE5 or GSE5-Like promoter sequence. These methods include both physical and chemical mutagenesis. A skilled person will know further approaches can be used to generate such mutants, and methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
In one embodiment, insertional mutagenesis is used, for example using T-DNA mutagenesis (which inserts pieces of the T-DNA from the Agrobacterium tumefaciens T-Plasmid into DNA causing either loss of gene function or gain of gene function mutations), site-directed nucleases (SDNs) or transposons as a mutagen. Insertional mutagenesis is an alternative means of disrupting gene function and is based on the insertion of foreign DNA into the gene of interest (see Krysan et al, The Plant Cell, Vol. 11, 2283-2290, December 1999). Accordingly, in one embodiment, T-DNA is used as an insertional mutagen to disrupt the GSE5 or GSE5-Like gene or GSE5 or GSE5-Like promoter expression. An example of using T-DNA mutagenesis to disrupt the Arabidopsis GSE5 gene is described in Downes et al. 2003. T-DNA not only disrupts the expression of the gene into which it is inserted, but also acts as a marker for subsequent identification of the mutation. Since the sequence of the inserted element is known, the gene in which the insertion has occurred can be recovered, using various cloning or PCR-based strategies. The insertion of a piece of T-DNA in the order of 5 to 25 kb in length generally produces a disruption of gene function. If a large enough population of T-DNA transformed lines is generated, there are reasonably good chances of finding a transgenic plant carrying a T-DNA insert within any gene of interest. Transformation of spores with T-DNA is achieved by an Agrobacterium-mediated method which involves exposing plant cells and tissues to a suspension of Agrobacterium cells.
The details of this method are well known to a skilled person. In short, plant transformation by Agrobacterium results in the integration into the nuclear genome of a sequence called T-DNA, which is carried on a bacterial plasmid. The use of T-DNA transformation leads to stable single insertions. Further mutant analysis of the resultant transformed lines is straightforward and each individual insertion line can be rapidly characterized by direct sequencing and analysis of DNA flanking the insertion. Gene expression in the mutant is compared to expression of the GSE5 or GSE5-Like nucleic acid sequence in a wild type plant and phenotypic analysis is also carried out.
In another embodiment, mutagenesis is physical mutagenesis, such as application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons or protons. The targeted population can then be screened to identify a GSE5 or GSE5-Like loss of function mutant.
In another embodiment of the various aspects of the invention, the method comprises mutagenizing a plant population with a mutagen. The mutagen may be a fast neutron irradiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (ENU), triethylmelamine (1′EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N′-nitro-Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy-6-chloro-9 [3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) or formaldehyde. Again, the targeted population can then be screened to identify a GSE5 or GSE5-Like gene or promoter mutant.
In another embodiment, the method used to create and analyse mutations is targeting induced local lesions in genomes (TILLING), reviewed in Henikoff et al, 2004. In this method, seeds are mutagenised with a chemical mutagen, for example EMS. The resulting M1 plants are self-fertilised and the M2 generation of individuals is used to prepare DNA samples for mutational screening. DNA samples are pooled and arrayed on microtiter plates and subjected to gene specific PCR. The PCR amplification products may be screened for mutations in the GSE5 or GSE5-Like target gene using any method that identifies heteroduplexes between wild type and mutant genes. For example, but not limited to, denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE), or by fragmentation using chemical cleavage. Preferably the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences. Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program. Any primer specific to the GSE5 or GSE5-Like nucleic acid sequence may be utilized to amplify the GSE5 or GSE5-Like nucleic acid sequence within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of the GSE5 or GSE5-Like gene where useful mutations are most likely to arise, specifically in the areas of the GSE5 or GSE5-Like gene that are highly conserved and/or confer activity as explained elsewhere. To facilitate detection of PCR products on a gel, the PCR primer may be labelled using any conventional labelling method. In an alternative embodiment, the method used to create and analyse mutations is EcoTILLING. EcoTILLING is molecular technique that is similar to TILLING, except that its objective is to uncover natural variation in a given population as opposed to induced mutations. The first publication of the EcoTILLING method was described in Comai et al. 2004.
Rapid high-throughput screening procedures thus allow the analysis of amplification products for identifying a mutation conferring the reduction or inactivation of the expression of the GSE5 or GSE5-Like gene as compared to a corresponding non-mutagenised wild type plant. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the target gene GSE5 or GSE5-Like. Loss of and reduced function mutants with increased seed size compared to a control can thus be identified.
Plants obtained or obtainable by such method which carry a functional mutation in the endogenous GSE5 or GSE5-Like gene or promoter locus are also within the scope of the invention
In an alternative embodiment, the expression of the GSE5 or GSE5-Like gene may be reduced at either the level of transcription or translation. For example, expression of a GSE5 or GSE5-Like nucleic acid or GSE5 or GSE5-Like promoter sequence, as defined herein, can be reduced or silenced using a number of gene silencing methods known to the skilled person, such as, but not limited to, the use of small interfering nucleic acids (siNA) against GSE5 or GSE5-Like. “Gene silencing” is a term generally used to refer to suppression of expression of a gene via sequence-specific interactions that are mediated by RNA molecules. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete “silencing” of expression.
In one embodiment, the siNA may include, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs and short hairpin RNA (shRNA) capable of mediating RNA interference.
The inhibition of expression and/or activity can be measured by determining the presence and/or amount of GSE5 or GSE5-Like transcript using techniques well known to the skilled person (such as Northern Blotting, RT-PCR and so on).
Transgenes may be used to suppress endogenous plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes and indicated by easily visible pigmentation changes. Subsequently it has been described how many, if not all plant genes can be “silenced” by transgenes. Gene silencing requires sequence similarity between the transgene and the gene that becomes silenced. This sequence homology may involve promoter regions or coding regions of the silenced target gene. When coding regions are involved, the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. It is likely that the various examples of gene silencing involve different mechanisms that are not well understood. In different examples there may be transcriptional or post-transcriptional gene silencing and both may be used according to the methods of the invention.
The mechanisms of gene silencing and their application in genetic engineering, which were first discovered in plants in the early 1990s and then shown in Caenorhabditis elegans are extensively described in the literature.
RNA-mediated gene suppression or RNA silencing according to the methods of the invention includes co-suppression wherein over-expression of the target sense RNA or mRNA, that is the GSE5 or GSE5-Like sense RNA or mRNA, leads to a reduction in the level of expression of the genes concerned. RNAs of the transgene and homologous endogenous gene are co-ordinately suppressed. Other techniques used in the methods of the invention include antisense RNA to reduce transcript levels of the endogenous target gene in a plant. In this method, RNA silencing does not affect the transcription of a gene locus, but only causes sequence-specific degradation of target mRNAs. An “antisense” nucleic acid sequence comprises a nucleotide sequence that is complementary to a “sense” nucleic acid sequence encoding a GSE5 or GSE5-Like protein, or a part of the protein, i.e. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA transcript sequence. The antisense nucleic acid sequence is preferably complementary to the endogenous GSE5 or GSE5-Like gene to be silenced. The complementarity may be located in the “coding region” and/or in the “non-coding region” of a gene. The term “coding region” refers to a region of the nucleotide sequence comprising codons that are translated into amino acid residues. The term “non-coding region” refers to 5′ and 3′ sequences that flank the coding region that are transcribed but not translated into amino acids (also referred to as 5′ and 3′ untranslated regions).
Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid sequence may be complementary to the entire GSE5 or GSE5-Like nucleic acid sequence as defined herein, but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5′ and 3′ UTR). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide. The length of a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less. An antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, an antisense nucleic acid sequence (e.g., an antisense oligonucleotide sequence) may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, e.g., phosphorothioate derivatives and acridine-substituted nucleotides may be used. Examples of modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art. The antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Preferably, production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.
The nucleic acid molecules used for silencing in the methods of the invention hybridize with or bind to mRNA transcripts and/or insert into genomic DNA encoding a polypeptide to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense nucleic acid sequences can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid sequences can also be delivered to cells using vectors.
RNA interference (RNAi) is another post-transcriptional gene-silencing phenomenon which may be used according to the methods of the invention. This is induced by double-stranded RNA in which mRNA that is homologous to the dsRNA is specifically degraded. It refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNA). The process of RNAi begins when the enzyme, DICER, encounters dsRNA and chops it into pieces called small-interfering RNAs (siRNA). This enzyme belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA.
Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. MicroRNAs (miRNAs) miRNAs are typically single stranded small RNAs typically 19-24 nucleotides long. Most plant miRNAs have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein.
miRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes. Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weiqelworld.orq).
Thus, according to the various aspects of the invention a plant may be transformed to introduce a RNAi, shRNA, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule that has been designed to target the expression of an GSE5 nucleic acid sequence and selectively decreases or inhibits the expression of the gene or stability of its transcript. Preferably, the RNAi, snRNA, dsRNA, shRNA siRNA, miRNA, amiRNA, ta-siRNA or cosuppression molecule used according to the various aspects of the invention comprises a fragment of at least 17 nt, preferably 22 to 26 nt and can be designed on the basis of the information shown in any of SEQ ID NOs:1 to 14 or 55 to 75. Guidelines for designing effective siRNAs are known to the skilled person. Briefly, a short fragment of the target gene sequence (e.g., 19-40 nucleotides in length) is chosen as the target sequence of the siRNA of the invention. The short fragment of target gene sequence is a fragment of the target gene mRNA. In preferred embodiments, the criteria for choosing a sequence fragment from the target gene mRNA to be a candidate siRNA molecule include 1) a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5′ or 3′ end of the native mRNA molecule, 2) a sequence from the target gene mRNA that has a G/C content of between 30% and 70%, most preferably around 50%, 3) a sequence from the target gene mRNA that does not contain repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT), 4) a sequence from the target gene mRNA that is accessible in the mRNA, 5) a sequence from the target gene mRNA that is unique to the target gene, 6) avoids regions within 75 bases of a start codon. The sequence fragment from the target gene mRNA may meet one or more of the criteria identified above. The selected gene is introduced as a nucleotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oligonucleotides. This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNAs. The output of this analysis is a score of possible siRNA oligonucleotides. The highest scores are used to design double stranded RNA oligonucleotides that are typically made by chemical synthesis. In addition to siRNA which is complementary to the mRNA target region, degenerate siRNA sequences may be used to target homologous regions. siRNAs according to the invention can be synthesized by any method known in the art. RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Additionally, siRNAs can be obtained from commercial RNA oligonucleotide synthesis suppliers.
siRNA molecules according to the aspects of the invention may be double stranded. In one embodiment, double stranded siRNA molecules comprise blunt ends. In another embodiment, double stranded siRNA molecules comprise overhanging nucleotides (e.g., 1-5 nucleotide overhangs, preferably 2 nucleotide overhangs). In some embodiments, the siRNA is a short hairpin RNA (shRNA); and the two strands of the siRNA molecule may be connected by a linker region (e.g., a nucleotide linker or a non-nucleotide linker). The siRNAs of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the siRNA. The skilled person will be aware of other types of chemical modification which may be incorporated into RNA molecules.
In one embodiment, recombinant DNA constructs as described in U.S. Pat. No. 6,635,805, incorporated herein by reference, may be used.
The silencing RNA molecule is introduced into the plant using conventional methods, for example a vector and Agrobacterium-mediated transformation. Stably transformed plants are generated and expression of the GSE5 or GSE5-Like gene compared to a wild type control plant is analysed.
Silencing of the GSE5 or GSE5-Like nucleic acid sequence may also be achieved using virus-induced gene silencing.
Thus, in one embodiment of the invention, the plant expresses a nucleic acid construct comprising a RNAi, shRNA snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or co-suppression molecule that targets the GSE5 or GSE5-Like nucleic acid sequence as described herein and reduces expression of the endogenous GSE5 or GSE5-Like nucleic acid sequence. A gene is targeted when, for example, the RNAi, snRNA, dsRNA, siRNA, shRNA miRNA, ta-siRNA, amiRNA or cosuppression molecule selectively decreases or inhibits the expression of the gene compared to a control plant. Alternatively, a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule targets a GSE5 or GSE5-Like nucleic acid sequence when the RNAi, shRNA snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule hybridises under stringent conditions to the gene transcript.
A further approach to gene silencing is by targeting nucleic acid sequences complementary to the regulatory region of the gene (e.g., the promoter and/or enhancers) of GSE5 or GSE5-Like to form triple helical structures that prevent transcription of the gene in target cells. Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. In particular, it can be envisaged that manmade molecules may be useful for inhibiting the biological function of a target polypeptide, or for interfering with the signalling pathway in which the target polypeptide is involved.
In one embodiment, the suppressor nucleic acids may be anti-sense suppressors of expression of the GSE5 or GSE5-Like polypeptides. In using anti-sense sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a “reverse orientation” such that transcription yields RNA which is complementary to normal mRNA transcribed from the “sense” strand of the target gene.
An anti-sense suppressor nucleic acid may comprise an anti-sense sequence of at least 10 nucleotides from the target nucleotide sequence. It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, although total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence.
The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expression to take place.
Suppressor nucleic acids may be operably linked to tissue-specific or inducible promoters. For example, integument and seed specific promoters can be used to specifically down-regulate a GSE5 or GSE5-Like nucleic acid in developing ovules and seeds to increase final seed size.
Nucleic acid which suppresses expression of a GSE5 or GSE5-Like polypeptide as described herein may be operably linked to a heterologous regulatory-sequence, such as a promoter, for example a constitutive, inducible, tissue-specific or developmental specific promoter. The construct or vector may be transformed into plant cells and expressed as described herein. Plant cells comprising such vectors are also within the scope of the invention.
In another aspect, the invention relates to a silencing construct obtainable or obtained by a method as described herein and to a plant cell comprising such construct.
Thus, aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and in a preferred embodiment exclude embodiments that are solely based on generating plants by traditional breeding methods.
In a further embodiment, the method may comprise reducing and/or abolishing the activity of GSE5 or GSE5-Like. In one example this may comprise reducing GSE5's ability to interact with calmodulin by mutating the IQ domain as described herein.
In another aspect, the invention extends to a plant obtained or obtainable by a method as described herein.
In a further aspect of the invention, there is provided a method of increasing cell proliferation in the spiklet hull of a plant, preferably in the grain-width direction, the method comprising reducing or abolishing the expression of at least one nucleic acid encoding a grain size on chromosome 5 (referred to herein as GSE5) or GSE5-Like polypeptide and/or reducing the activity of a GSE5 or GSE5-Like polypeptide in said plant. The terms “increase”, “improve” or “enhance” as used herein are interchangeable. In one embodiment, cell proliferation is increased by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40% or 50% in comparison to a control plant.
In another aspect of the invention there is provided a genetically altered plant, part thereof or plant cell characterised in that the plant does not express GSE5 or GSE5-Like, has reduced levels of GSE5 or GSE5-Like expression, does not express a functional GSE5 or GSE5-Like protein or expresses a GSE5 or GSE5-Like protein with reduced function and/or activity. For example, the plant is a reduction (knock down) or loss of function (knock out) mutant wherein the function of the GSE5 or GSE5-Like nucleic acid sequence is reduced or lost compared to a wild type control plant. To this end, a mutation is introduced into either the GSE5 or GSE5-Like gene sequence or the corresponding promoter sequence which disrupts the transcription of the gene. Therefore, preferably said plant comprises at least one mutation in the promoter and/or gene for GSE5 and/or GSE5-Like. In one embodiment the plant may comprise a mutation in both the promoter and gene for GSE5 or GSE5-Like.
In a further aspect of the invention, there is provided a plant, part thereof or plant cell characterised by an increased seed yield compared to a wild-type or control pant, wherein preferably, the plant comprises at least one mutation in the GSE5 or GSE5-Like gene and/or its promoter. Preferably said increase in seed yield comprises an increase in at least one of seed weight, seed width and TKW.
The plant may be produced by introducing a mutation, preferably a deletion, insertion or substitution into the GSE5 or GSE5-Like gene and/or promoter sequence by any of the above described methods. Preferably said mutation is introduced into a least one plant cell and a plant regenerated from the at least one mutated plant cell.
Alternatively, the plant or plant cell may comprise a nucleic acid construct expressing an RNAi molecule targeting the GSE or GSE5-Like gene as described herein. In one embodiment, said construct is stably incorporated into the plant genome. These techniques also include gene targeting using vectors that target the gene of interest and which allow integration of a transgene at a specific site. The targeting construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene will be translated into a nonfunctional protein, if it is translated at all.
In another aspect of the invention there is provided a method for producing a genetically altered plant as described herein. In one embodiment, the method comprises introducing at least one mutation into the GSE5 or GSE5-Like gene and/or GSE5 or GSE5-Like promoter of preferably at least one plant cell using any mutagenesis technique described herein. Preferably said method further comprising regenerating a plant from the mutated plant cell.
The method may further comprise selecting one or more mutated plants, preferably for further propagation. Preferably said selected plants comprise at least one mutation in the GSE5 or GSE5-Like gene and/or promoter sequence. Preferably said plants are characterised by abolished or a reduced level of GSE5 or GSE5-Like expression and/or a reduced level of GSE5 or GSE5-Like polypeptide activity. Expression and/or activity levels of GSE5 or GSE5-Like can be measured by any standard technique known to the skilled person. In one embodiment GSE5 binding to calmodulin could be measured. A reduction is as described herein.
The selected plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
In a further aspect of the invention there is provided a plant obtained or obtainable by the above described methods.
For the purposes of the invention, a “genetically altered plant” or “mutant plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant. In one embodiment, a mutant plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as any of the mutagenesis methods described herein. In one embodiment, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, the plant genome has been altered compared to wild type sequences using a mutagenesis method. Such plants have an altered phenotype as described herein, such as an increased seed yield. Therefore, in this example, increased seed yield is conferred by the presence of an altered plant genome, for example, a mutated endogenous GSE5 or GSE5-Like gene or GSE5 or GSE5-Like promoter sequence. In one embodiment, the endogenous promoter or gene sequence is specifically targeted using targeted genome modification and the presence of a mutated gene or promoter sequence is not conferred by the presence of transgenes expressed in the plant. In other words, the genetically altered plant can be described as transgene-free.
A plant according to the various aspects of the invention, including the transgenic plants, methods and uses described herein may be a monocot or a dicot plant. Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal. In another embodiment the plant is Arabidopsis or Medicago truncatula.
In a most preferred embodiment, the plant is selected from rice, wheat, maize, soybean and sorghum. In a most preferred embodiment the plant is rice, preferably the japonica or indica varieties.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned comprise the nucleic acid construct as described herein. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the nucleic acid construct as described herein.
The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. Another product that may derived from the harvestable parts of the plant of the invention is biodiesel. The invention also relates to food products and food supplements comprising the plant of the invention or parts thereof. In one embodiment, the food products may be animal feed. In another aspect of the invention, there is provided a product derived from a plant as described herein or from a part thereof.
In a most preferred embodiment, the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed produced from a genetically altered plant as described herein. In an alternative embodiment, the plant part is pollen, a propagule or progeny of the genetically altered plant described herein. Accordingly, in a further aspect of the invention there is provided pollen, a propagule or progeny of the genetically altered plant as described herein.
A control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. Accordingly, in one embodiment, the control plant does not have reduced expression of a GSE5 or GSE5-Like nucleic acid and/or reduced activity of a GSE5 or GSE5-Like polypeptide. In an alternative embodiment, the plant been genetically modified, as described above. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.
Genome Editing Constructs for Use with the Methods for Targeted Genome Modification Described Herein
By “crRNA” or CRISPR RNA is meant the sequence of RNA that contains the protospacer element and additional nucleotides that are complementary to the tracrRNA.
By “tracrRNA” (transactivating RNA) is meant the sequence of RNA that hybridises to the crRNA and binds a CRISPR enzyme, such as Cas9 thereby activating the nuclease complex to introduce double-stranded breaks at specific sites within the genomic sequence of at least one GSE5 or GSE5-Like nucleic acid or promoter sequence.
By “protospacer element” is meant the portion of crRNA (or sgRNA) that is complementary to the genomic DNA target sequence, usually around 20 nucleotides in length. This may also be known as a spacer or targeting sequence.
By “sgRNA” (single-guide RNA) is meant the combination of tracrRNA and crRNA in a single RNA molecule, preferably also including a linker loop (that links the tracrRNA and crRNA into a single molecule). “sgRNA” may also be referred to as “gRNA” and in the present context, the terms are interchangeable. The sgRNA or gRNA provide both targeting specificity and scaffolding/binding ability for a Cas nuclease. A gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule.
By “TAL effector” (transcription activator-like (TAL) effector) or TALE is meant a protein sequence that can bind the genomic DNA target sequence (a sequence within the GSE5 or GSE5-Like gene or promoter sequence) and that can be fused to the cleavage domain of an endonuclease such as Fokl to create TAL effector nucleases or TALENS or meganucleases to create megaTALs. A TALE protein is composed of a central domain that is responsible for DNA binding, a nuclear-localisation signal and a domain that activates target gene transcription. The DNA-binding domain consists of monomers and each monomer can bind one nucleotide in the target nucleotide sequence. Monomers are tandem repeats of 33-35 amino acids, of which the two amino acids located at positions 12 and 13 are highly variable (repeat variable diresidue, RVD). It is the RVDs that are responsible for the recognition of a single specific nucleotide. HD targets cytosine; NI targets adenine, NG targets thymine and NN targets guanine (although NN can also bind to adenine with lower specificity).
In another aspect of the invention there is provided a nucleic acid construct wherein the nucleic acid construct encodes at least one DNA-binding domain, wherein the DNA-binding domain can bind to a sequence in the GSE5 gene or GSE5-Like gene, wherein said sequence is selected from SEQ ID NOs: 15 to 20, 48, 51, 76, 79, 80, 81, 82, 83 and 84. In one embodiment, said construct further comprises a nucleic acid encoding a SSN, such as Fokl or a Cas protein.
In one embodiment, the nucleic acid construct encodes at least one protospacer element wherein the sequence of the protospacer element is selected from SEQ ID NOs: 21 to 26 or 52 or 77 or a variant thereof.
In a further embodiment, the nucleic acid construct comprises a crRNA-encoding sequence. As defined above, a crRNA sequence may comprise the protospacer elements as defined above and preferably additional nucleotides that are complementary to the tracrRNA. An appropriate sequence for the additional nucleotides will be known to the skilled person as these are defined by the choice of Cas protein.
In another embodiment, the nucleic acid construct further comprises a tracrRNA sequence. Again, an appropriate tracrRNA sequence would be known to the skilled person as this sequence is defined by the choice of Cas protein.
In a further embodiment, the nucleic acid construct comprises at least one nucleic acid sequence that encodes a sgRNA (or gRNA). Again, as already discussed, sgRNA typically comprises a crRNA sequence, a tracrRNA sequence and preferably a sequence for a linker loop. In a preferred embodiment, the nucleic acid construct comprises at least one nucleic acid sequence that encodes a sgRNA sequence as defined herein in SEQ ID NO: 78 or variant thereof.
In a further embodiment, the nucleic acid construct may further comprise at least one nucleic acid sequence encoding an endoribonuclease cleavage site. Preferably the endoribonuclease is Csy4 (also known as Cas6f). Where the nucleic acid construct comprises multiple sgRNA nucleic acid sequences the construct may comprise the same number of endoribonuclease cleavage sites. In another embodiment, the cleavage site is 5′ of the sgRNA nucleic acid sequence. Accordingly, each sgRNA nucleic acid sequence is flanked by a endoribonuclease cleavage site.
The term ‘variant’ refers to a nucleotide sequence where the nucleotides are substantially identical to one of the above sequences. The variant may be achieved by modifications such as an insertion, substitution or deletion of one or more nucleotides. In a preferred embodiment, the variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of the above sequences. In one embodiment, sequence identity is at least 90%. In another embodiment, sequence identity is 100%. Sequence identity can be determined by any one known sequence alignment program in the art.
The invention also relates to a nucleic acid construct comprising a nucleic acid sequence operably linked to a suitable plant promoter. A suitable plant promoter may be a constitutive or strong promoter or may be a tissue-specific promoter. In one embodiment, suitable plant promoters are selected from, but not limited to U3 and U6.
The nucleic acid construct of the present invention may also further comprise a nucleic acid sequence that encodes a CRISPR enzyme. By “CRISPR enzyme” is meant an RNA-guided DNA endonuclease that can associate with the CRISPR system. Specifically, such an enzyme binds to the tracrRNA sequence. In one embodiment, the CRIPSR enzyme is a Cas protein (“CRISPR associated protein), preferably Cas 9 or Cpf1, more preferably Cas9. In a specific embodiment Cas9 is a codon-optimised Cas9 (specific for the plant in question). In one embodiment, Cas9 has the sequence described in SEQ ID NO: 33 or a functional variant or homolog thereof. In another embodiment, the CRISPR enzyme is a protein from the family of Class 2 candidate x proteins, such as C2c1, C2C2 and/or C2c3. In one embodiment, the Cas protein is from Streptococcus pyogenes. In an alternative embodiment, the Cas protein may be from any one of Staphylococcus aureus, Neisseria meningitides, Streptococcus thermophiles or Treponema denticola.
The term “functional variant” as used herein with reference to Cas9 refers to a variant Cas9 gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence, for example, acts as a DNA endonuclease, or recognition or/and binding to DNA. A functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. In one embodiment, a functional variant of SEQ ID NO: 33 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acid represented by SEQ ID NO: 33. In a further embodiment, the Cas9 protein has been modified to improve activity.
Suitable homologs or orthologs can be identified by sequence comparisons and identifications of conserved domains. The function of the homolog or ortholog can be identified as described herein and a skilled person would thus be able to confirm the function when expressed in a plant.
In an alternative aspect of the invention, the nucleic acid construct comprises at least one nucleic acid sequence that encodes a TAL effector, wherein said effector targets a GSE5 sequence selected from SEQ ID NOs: 15 to 20 or 48 or 51 or a GSE5-Like sequence selected from SEQ ID NOs 76 and 79 to 84. Methods for designing a TAL effector would be well known to the skilled person, given the target sequence. Examples of suitable methods are given in Sanjana et al., and Cermak T et al, both incorporated herein by reference. Preferably, said nucleic acid construct comprises two nucleic acid sequences encoding a TAL effector, to produce a TALEN pair. In a further embodiment, the nucleic acid construct further comprises a sequence-specific nuclease (SSN). Preferably such SSN is a endonuclease such as Fokl. In a further embodiment, the TALENs are assembled by the Golden Gate cloning method in a single plasmid or nucleic acid construct.
In another aspect of the invention, there is provided a sgRNA molecule, wherein the sgRNA molecule comprises a crRNA sequence and a tracrRNA sequence and wherein the crRNA sequence can bind to at least one sequence selected from SEQ ID NOs: 15 to 20, 48, 51, 76 or 79 to 84 or a variant thereof.
A “variant” is as defined herein. In one embodiment, the sgRNA molecule may comprise at least one chemical modification, for example that enhances its stability and/or binding affinity to the target sequence or the crRNA sequence to the tracrRNA sequence. Such modifications would be well known to the skilled person, and include for example, but not limited to, the modifications described in Randar et al., 2015, incorporated herein by reference. In this example the crRNA may comprise a phosphorothioate backbone modification, such as 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me) and S-constrained ethyl (cET) substitutions.
In another aspect of the invention, there is provided an isolated nucleic acid sequence that encodes for a protospacer element (as defined in any of SEQ ID NOs: 21 to 26 or 52 or 77), or a sgRNA.
In another aspect of the invention, there is provided a plant or part thereof or at least one isolated plant cell transfected with at least one nucleic acid construct as described herein. Cas9 and sgRNA may be combined or in separate expression vectors (or nucleic acid constructs, such terms are used interchangeably). In other words, in one embodiment, an isolated plant cell is transfected with a single nucleic acid construct comprising both sgRNA and Cas9 as described in detail above. In an alternative embodiment, an isolated plant cell is transfected with two nucleic acid constructs, a first nucleic acid construct comprising at least one sgRNA as defined above and a second nucleic acid construct comprising Cas9 or a functional variant or homolog thereof. The second nucleic acid construct may be transfected below, after or concurrently with the first nucleic acid construct. The advantage of a separate, second construct comprising a cas protein is that the nucleic acid construct encoding at least one sgRNA can be paired with any type of cas protein, as described herein, and therefore is not limited to a single cas function (as would be the case when both cas and sgRNA are encoded on the same nucleic acid construct).
In one embodiment, the nucleic acid construct comprising a cas protein is transfected first and is stably incorporated into the genome, before the second transfection with a nucleic acid construct comprising at least one sgRNA nucleic acid. In an alternative embodiment, a plant or part thereof or at least one isolated plant cell is transfected with mRNA encoding a cas protein and co-transfected with at least one nucleic acid construct as defined herein.
Cas9 expression vectors for use in the present invention can be constructed as described in the art. In one example, the expression vector comprises a nucleic acid sequence as defined herein or a functional variant or homolog thereof, wherein said nucleic acid sequence is operably linked to a suitable promoter. Examples of suitable promoters include, but are not limited to Cas9, 35S and Actin.
In an alternative aspect of the present invention, there is provided an isolated plant cell transfected with at least one sgRNA molecule as described herein.
In a further aspect of the invention, there is provided a genetically modified or edited plant comprising the transfected cell described herein. In one embodiment, the nucleic acid construct or constructs may be integrated in a stable form. In an alternative embodiment, the nucleic acid construct or constructs are not integrated (i.e. are transiently expressed). Accordingly, in a preferred embodiment, the genetically modified plant is free of any sgRNA and/or Cas protein nucleic acid. In other words, the plant is transgene free.
The term “introduction”, “transfection” or “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art. The transfer of foreign genes into the genome of a plant is called transformation.
Transformation of plants is now a routine technique in many species. Any of several transformation methods known to the skilled person may be used to introduce the nucleic acid construct or sgRNA molecule of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation.
Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant (microinjection), gene guns (or biolistic particle delivery systems (bioloistics)) as described in the examples, lipofection, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, ultrasound-mediated gene transfection, optical or laser transfection, transfection using silicon carbide fibers, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, can also be produced via Agrobacterium tumefaciens mediated transformation, including but not limited to using the floral dip/Agrobacterium vacuum infiltration method as described in Clough & Bent (1998) and incorporated herein by reference.
Accordingly, in one embodiment, at least one nucleic acid construct or sgRNA molecule as described herein can be introduced to at least one plant cell using any of the above described methods. In an alternative embodiment, any of the nucleic acid constructs described herein may be first transcribed to form a preassembled Cas9-sgRNA ribonucleoprotein and then delivered to at least one plant cell using any of the above described methods, such as lipofection, electroporation or microinjection.
Optionally, to select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. As described in the examples, a suitable marker can be bar-phosphinothricin or PPT. Alternatively, the transformed plants are screened for the presence of a selectable marker, such as, but not limited to, GFP, GUS (β-glucuronidase). Other examples would be readily known to the skilled person. Alternatively, no selection is performed, and the seeds obtained in the above-described manner are planted and grown and GSE5 expression or protein levels measured at an appropriate time using standard techniques in the art. This alternative, which avoids the introduction of transgenes, is preferable to produce transgene-free plants.
Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using PCR to detect the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, integration and expression levels of the newly introduced DNA may be monitored using Southern, Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
In a further related aspect of the invention, there is also provided, a method of obtaining a genetically modified plant as described herein, the method comprising
In a further embodiment, the method also comprises the step of screening the genetically modified plant for SSN (preferably CRISPR)-induced mutations in the GSE5 gene or promoter sequence. In one embodiment, the method comprises obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect a mutation in at least one GSE5 or GSE5-Like gene or promoter sequence.
In a further embodiment, the methods comprise generating stable T2 plants preferably homozygous for the mutation (that is a mutation in at least one GSE5 or GSE5-Like gene or promoter sequence).
Plants that have a mutation in at least one GSE5 or GSE5-Like gene and/or promoter sequence can also be crossed with another plant also containing at least one mutation in at least one GSE5 or GSE5-Like gene and/or promoter sequence to obtain plants with additional mutations in the GSE5 gene or GSE5-Like or promoter sequence. The combinations will be apparent to the skilled person. Accordingly, this method can be used to generate a T2 plants with mutations on all or an increased number of homoeologs, when compared to the number of homoeolog mutations in a single T1 plant transformed as described above.
A plant obtained or obtainable by the methods described above is also within the scope of the invention.
A genetically altered plant of the present invention may also be obtained by transference of any of the sequences of the invention by crossing, e.g., using pollen of the genetically altered plant described herein to pollinate a wild-type or control plant, or pollinating the gynoecia of plants described herein with other pollen that does not contain a mutation in at least one of the GSE5 or GSE5-Like gene or promoter sequence. The methods for obtaining the plant of the invention are not exclusively limited to those described in this paragraph; for example, genetic transformation of germ cells from the ear of wheat could be carried out as mentioned, but without having to regenerate a plant afterward.
In a further aspect of the invention, there is provided a method for screening a population of plants and identifying and/or selecting a plant that will have reduced GSE5 or GSE5-Like expression and/or an increased seed yield phenotype, preferably an increased seed width, weight or TKW, the method comprising detecting in the plant or plant germplasm at least one polymorphism (preferably a low GSE5 or GSE5-Like expresser polymorphism) in the promoter of the GSE5 or GSE5-Like gene. Preferably, said screening comprises determining the presence of at least one polymorphism, wherein said polymorphism is at least one insertion and/or at least one deletion.
In one embodiment, a plant expressing a deletion of a nucleic acid sequence comprising SEQ ID NO: 30 will express ˜0.6 fold lower level of GSE5 expression compared to a plant wherein the promoter without this polymorphism. In one embodiment, the plant is rice, preferably the japonica variety. Such plants are referred to herein as GSE5DEL2.
In another embodiment, a plant expressing a deletion of a nucleic acid sequence comprising SEQ ID NO: 29 and/or the insertion of a nucleic acid sequence comprising SEQ ID NO: 31 will express ˜0.65 fold lower level of GSE5 expression compared to a plant wherein the promoter without this polymorphism. In one embodiment, the plant is rice, preferably the indica variety. Such plants are referred to herein as GSE5DEL1+IN1.
As a result, the above-described plants will display an increased seed yield as described above.
Suitable tests for assessing the presence of a polymorphism would be well known to the skilled person, and include but are not limited to, Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs-which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs). In one embodiment, Kompetitive Allele Specific PCR (KASP) genotyping is used.
In one embodiment, the method comprises
a) obtaining a nucleic acid sample from a plant and
b) carrying out nucleic acid amplification of one or more GSE5 promoter alleles using one or more primer pairs.
In a further embodiment, the method may further comprise introgressing the chromosomal region comprising at least one of said low-GSE5-expressing polymorphisms or the chromosomal region containing the repeat sequence deletion as described above into a second plant or plant germplasm to produce an introgressed plant or plant germplasm. Preferably the expression of GSE5 or GSE5-Like in said second plant will be reduced or abolished, and more preferably said second plant will display an increase in seed size, and increase in total protein and/or lipid content and/or a reduction in glucosinolate levels.
In one embodiment, plants of the GSE5DEL2 and GSE5DEL1+IN1 haplotypes may be selected and the levels of GSE5 nucleic acid and/or activity of the GSE5 protein reduced or further reduced by any method described herein.
Accordingly, in a further aspect of the invention there is provided a method for increasing yield, preferably seed or grain yield in a plant, the method comprising
By “further reducing” is meant reducing the level of GSE5 expression to a level lower than that in the plant with the GSE5DEL2 and GSE5DEL1+IN1 haplotype in step a. The terms “reducing” means a decrease in the levels of GSE5 expression and/or activity by up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a GSE5DEL2 and GSE5DEL1+IN1 control plant.
The inventors have also surprisingly identified that increasing the expression of GSE5 or GSE5-Like results in slender grains—i.e. an increase in grain length.
The terms “increase”, “improve” or “enhance” as used herein are interchangeable. In one embodiment, grain length is increased by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 120% or more in comparison to a control plant. Preferably, the increase is at least 2-10%, more preferably 3-8%.
Accordingly, in another aspect of the invention there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide as defined in SEQ ID NO: 1 or 57 or a functional variant or homolog thereof, wherein said sequence is operably linked to a regulatory sequence, wherein preferably said regulatory sequence is a tissue-specific promoter or a constitutive promoter. In a further embodiment, the nucleic acid construct comprises a nucleic acid sequence as defined in SEQ ID NO: 2 or 56 (cDNA) or 32 or 55 (genomic) or a functional variant or homolog thereof. A functional variant or homolog is as defined above.
The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
A “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The “plant promoter” can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other “plant” regulatory signals, such as “plant” terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3′-regulatory region such as terminators or other 3′ regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern. The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
In one embodiment, the promoter is a constitutive promoter. A “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include but are not limited to actin, HMGP, CaMV19S, GOS2, rice cyclophilin, maize H3 histone, alfalfa H3 histone, 34S FMV, rubisco small subunit, OCS, SAD1, SAD2, nos, V-ATPase, super promoter, G-box proteins and synthetic promoters.
In another aspect of the invention there is provided a vector comprising the nucleic acid sequence described above.
In a further aspect of the invention, there is provided a host cell comprising the nucleic acid construct. The host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell. The invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described below.
In another embodiment, there is provided a transgenic plant expressing the nucleic acid construct as described above. In one embodiment, said nucleic acid construct is stably incorporated into the plant genome.
The nucleic acid sequence is introduced into said plant through a process called transformation as described above.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
A suitable plant is defined above.
In another aspect, the invention relates to the use of a nucleic acid construct as described herein to increase grain length as defined above.
In a further aspect of the invention there is provided a method of increasing grain length, the method comprising introducing and expressing in said plant the nucleic acid construct described herein.
In another aspect of the invention there is provided a method of producing a plant with an increased grain length the method comprising introducing and expressing in said plant the nucleic acid construct described herein.
Said increase is relative to a control or wild-type plant.
While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The invention is now described in the following non-limiting example.
The utilization of natural genetic variation greatly contributes to improvement of important agronomic traits in crops. Understanding the genetic basis for natural variation of grain size can help breeders develop high-yield rice varieties. Here we identify a novel quantitative trait locus for grain size (GSE5) using a genome-wide association study (GWAS) with functional testing. GSE5 encodes a plasma membrane-associated protein with IQ domains (IQD), which associates with calmodulin (OsCaM1-1). GSE5 regulates grain size by influencing cell proliferation. We identify three major haplotypes (GSE5, GSE5DEL1+IN1 and GSE5DEL2) in cultivated rice according to the deletion/insertion type in the promoter of GSE5. We demonstrate that the deletion 1 (DEL1) in indica varieties carrying the GSE5DEL1+IN1 haplotype and the deletion 2 (DEL2) in japonica varieties carrying the GSE5DEL2 haplotype cause the decreased expression of GSE5, resulting in wide grains. We generate loss-of-function mutant of GSE5 that increases grain width and weight, while overexpression of GSE5 results in slender grains. Further analyses indicate that wild rice accessions contain GSE5, GSE5DEL1+IN1 and GSE5DEL2 haplotypes, suggesting that these three major haplotypes in cultivated rice are likely to have originated from different wild rice accessions during rice domestication. Thus, these findings identify a novel QTL gene for grain size (GSE5) that is widely utilized by rice breeders and reveal that natural variation in the promoter of GSE5 contributes to grain size diversity in rice.
To identify natural variation in genes involved in grain size control, we performed the genome-wide association study (GWAS) with functional analysis. We used 102 indica varieties, which showed large variation in grain size (
We performed GWAS for grain width in this indica population using a mixed linear model with correction of kinship, which is a widely used method for GWAS analysis (Huang et al., 2010; Yano et al., 2016). As shown in
Expression Level of LOC_Os05g09520 is Associated with Grain Width
To identify the GSE5 gene, we used pairwise LD correlations (r2>0.6) (Yano et al., 2016) to estimate a candidate region from 5.357 Mb to 5.379 Mb (22.42 kb) (
To understand why expression of LOC_Os05g09520 is decreased in wide grain varieties, we examined the 5′-flanking sequences of LOC_Os05g09520 in indica varieties and found that most wide grain indica varieties contain a 950-bp deletion (DEL1) as well as a 367-bp insertion (IN1) (
As the japonica varieties had the 1212-bp deletion (DEL2) that partially overlaps with DEL1 (
To determine whether DEL1 and IN1 in indica varieties and DEL2 in japonica varieties could decrease expression of LOC_Os05g09520, we investigated the activity of the promoter (proGSE5) without or with DEL1 and IN1 (proGSE5DEL1+IN1), the only DEL1 (proGSE5DEL1) and the DEL2 (proGSE5DEL2), respectively (
To confirm that LOC_Os05g09520 is the GSE5 gene, we generated the loss-of-function mutant for LOC_Os05g09520 and performed a genetic complementation test.
The japonica variety Zhonghua 11 (ZH11) with the deletion DEL2 in the promoter of LOC_Os05g09520 had wide grains. Although the ZH11 promoter (proGSE5DEL2) had reduced activity, it still possessed partial activity (
The spikelet hull restricts the growth of a grain, which has been proposed to influence grain size in rice (Li and Li, 2016). Cell proliferation and cell expansion coordinately determine the growth of spikelet hulls. We therefore measured cell number and cell size in ZH11 and GSE5-cr spikelet hulls. The GSE5-cr spikelet hulls contained more epidermal cells than ZH11 spikelet hulls in the grain-width direction (
We then investigated cell number and cell size in ZH11 and proActin:GSE5 spikelet hulls. As shown in
GSE5 Encodes a Plasma Membrane-Associated Protein with IQ Domains (IQD)
Grain size and weight are important agronomic traits in crops. We identify a novel grain size gene (GSE5) that encodes a plasma membrane-associated protein with IQ domains (IQD), which interacts with calmodulin (OsCaM1-1). In rice, loss of GSE5 function causes wide and heavy grains, while overexpression of GSE5 results in narrow and long grains. By performing a BAST search in the databases, we found that GSE5 shares significantly similarity with its homologs in other crops, such as maize, wheat, sorghum and brachypodium. Our current knowledge of GSE5 functions suggest that GSE5 and its homologs in other crops or plant species could be used to engineer large and heavy seeds in these key crops. We could use CRISPR/Cas9 technology to knock-out GSE5 or its homologs in other crops to increase seed size and weight in these crops. We also could use RNAi technology to knock-down the expression of GSE5 or its homologs in crops to increase seed size and weight in these crops.
GSE5 encodes a predicted protein with IQ domains (IQD) (
GSE5 transcripts were detected in developing panicles using quantitative real-time RT-PCR analysis (
To determine the subcellular localization of GSE5, we expressed a GSE5-GFP fusion protein under its own promoter (proGSE5:GSE5-GFP) in the japonica variety ZH11. The proGSE5:GSE5-GFP transgenic plants produced narrow grains compared with ZH11 (
Based on the deletion/insertion type in the promoter of GSE5, we identified three major haplotypes (GSE5, GSE5DEL1+IN1 and GSE5DEL2) in cultivated rice (
Cultivated rice has been proposed to be domesticated from wild rice (Oryza rufipogon). We therefore asked whether wild rice accessions could contain these two deletions (DEL1 and DEL2) in the promoter region of GSE5. We genotyped 41 wild rice accessions (O. rufipogon) and observed that most accessions had the GSE5 haplotype, five accessions contained the GSE5DEL1+IN1 haplotype, and only one wild rice accession that came from Hunan province in the south region of China had the GSE5DEL2 haplotype (
In summary, our findings identify a novel quantitative trait gene for grain size (GSE5) using a genome-wide association study with functional testing, which is widely utilized by rice breeders. We demonstrate that natural variation in the promoter of GSE5 contributes to grain size diversity in cultivated rice. Our findings provide insight into the genetic basis for natural variation in rice grain size control.
The cultivated rice varieties were obtained from a collection of cultivated rice preserved at the China National Rice Research Institute. The common wild rice varieties (Oryza rufipogon) were obtained from the Institute of Botany, Chinese Academy of Sciences (Zheng and Ge, 2010; Zhu et al., 2007). The indica and japonica varieties used in this study were cultivated in the paddy fields at Hangzhou (China) and Hainan (China).
Grain size of the 102 indica varieties was measured using the SC Detection and Analysis System of Rice Seeds (Hangzhou WSeen Detection Technology). Dry grains of Zhonghua 11 (ZH11) and GSE5-cr were weighted using electronic analytical balance (METTLER MOLEDO AL104 CHINA).
To observe cell size and cell number, grain hulls of Zhonghua 11 (ZH11), GSE5-cr and proActin:GSE5 transgenic plants were sputter-coated with platinum and observed using a scanning electron microscope (SEM) (HITACHI S-3000N). Image J software was used to measure epidermal cell size.
NuClean PlantGen DNA kits (CWBIO, China) were used for the genomic DNA extraction. For each cultivated rice, a single individual was used for genome sequencing on the Illumina Hiseq 2500. Library construction and sample indexing were performed as described previously (Huang et al., 2009). The libraries were loaded into the Illumina Hiseq 2500 for 100 bp paired-end sequencing. Image analysis and base calling were conducted using the Illumina Genome Analyzer processing pipeline (v1.4). PERL scripts in the SEG-Map pipeline were used to sort raw sequences on the basis of the 5′ indexes.
A total of 6.773×109 paired-end 100-bp reads were obtained for the cultivated accessions. Firstly, quality control was performed, and the average Q30 was 89.94%, which means that the reads were reliable. Then the reads were aligned to Os-Nipponbare-Reference-MSU7.0 pseudomolecules using bwa-mem with the -M option of BWA software (Li and Durbin, 2010). The mapped reads were realigned using RealignerTargetCreator and indelRealigner of GATK software (DePristo et al., 2011). To label SNPs, UnifiedGenotyper of GATK was used with the -glm BOTH option. All nucleotide polymorphisms were analyzed according to their location in the reference genome.
The population structure of the 102 indica varieties (PCA) was estimated using the software PLINK version 1.9 (http://pngu.mgh.harvard.edu/-purcell/plink/). The LD between SNPs in the 102 varieties was evaluated using squared Pearson's correlation coefficient (r2) as calculated with the -r2 command in the software PLINK version 1.9. The LD heatmaps surrounding peaks in the GWAS were constructed using the R package “LD heatmap” (Shin et al., 2006). We estimated the candidate regions using an r2>0.6 (Yano et al., 2016).
Genome wide association study (GWAS)
The population structure (Q) was inferred using Admixture (Alexander et al., 2009), and the best one was selected when cross-validation (CV) errors was minimum. The relative kinship matrix (K) of the natural population was calculated using TASSEL 5.2.1 (Bradbury et al., 2007). GWAS was performed using the Q+K model in TASSEL 5.2.1. The genome-wide significance threshold was determined using permutation-based false-discovery-rate-adjusted P values (Dudbridge and Gusnanto, 2008). The permutation tests were repeated 1,000 times.
The 7897-bp GSE5 genomic sequence was amplified from the indica variety 93-11 using the primers gGUS-F/R and gGFP-F/R and cloned into the pMDC164 and pMDC107 vectors using in-fusion enzyme (Genebank Biosciences Inc, China), respectively. The coding sequences of GSE5 and GSE5L1 were amplified by the specific primers cGSE5-F/R and cGSE5L1-F/R and cloned into the plpkb003 vector using in-fusion enzyme (Genebank Biosciences Inc, China) to generate proActin:GSE5 and proActin:GSE5L1 plasmids, respectively. The 488-bp sequence was amplified from the PCR products of crGSE5-1 and crGSE5-2 using the primers crGSE5-1F and crGSE5-2R and cloned into the vector pMDC99-Cas9 using in-fusion enzyme (Genebank Biosciences Inc, China) to generate the CRISPR/Cas9-GSE5 plasmid. The plasmids were introduced into Agrobaterium tumefaciens strain GV3101 by electroporation, and rice transformation was transformed according to a previous published method (Hiei et al., 1994).
The developing panicles of proGSE5:GSE5-GUS transgenic plants were stained in a GUS buffer according to the method described previously (Wang et al., 2016). The roots of proGSE5:GSE5-GFP transgenic plants were used to investigate the subcellular localization of GSE5. Plasma membrane were stained using FM4-64 (5 μg/ml), and samples were observed using Zeiss LSM 710 NLO confocal microscopy.
The coding sequence of GSE5 were amplified by specific primers ycGSE5-F/R, fused with the C-terminal fragment of YFP (cYFP), and then subcloned into the pGWB414 vector (Invitrogen) using in-fusion enzyme (Genebank Biosciences Inc, China). The N-terminal fragment of YFP (nYFP) was amplified from pSY736 using the primers YN-736-F and YN-736-R, fused with the OsCaM1-1 gene, and then subcloned into the pGWB414 vector (Invitrogen) using in-fusion enzyme (Genebank Biosciences Inc, China). nYFP-OsCaM1-1 and cYFP-GSE5 constructs were transformed into Agrobacterium strains GV3101. Transient expression of nYFP-OsCaM1-1 and cYFP-GSE5 in Nicotiana benthamiana leaves and fluorescence observation were conducted as described previously (Wang et al., 2016).
Developing panicles were used to extract total RNA using an RNAprep pure Plant Kit (TIANGEN, China). Total RNA was used for cDNA synthesis with SuperScript III Reverse Transcriptase (Invitrogen). A Lightcycler 480 machine (Roche) was used to conduct quantitative real-time PCR. Relative amounts of qSW5 and GSE5 were calculated using the comparative threshold (Wang et al., 2016). The primers for quantitative real-time RT-PCR are shown in Supplementary Table 4.
The promoter sequences of 6320-bp, 5310-bp and 4547-bp were amplified from indica variety 93-11 genomic DNA using the specific primers of pLUCL-F/R, pLUCM-F/R and pLUCS-F/R and constructed into the vector pGreenII0800-LUC (Hellens et al., 2005) to generate proGSE5:LUC, proGSE5DEL1:LUC and proGSE5DEL2:LUC plasmids, respectively. For proGSE5DEL1+IN1:LUC construction, the 5677-bp PCR fragment was amplified from indica variety Zhefu802 using the specific primers pLUCM-F/R and cloned into the vector pGreenII0800-LUC using in-fusion enzyme (Genebank Biosciences Inc, China). The plasmids were transferred into the Agrobaterium tumefaciens strain GV3101 by electroporation and coinfiltrated into Nicotiana benthamiana leaves. The Firefly and Renilla luciferase activities were measured using a Dual-Luciferase® Reporter Assay System (Promega).
To analyse the evolutionary history, the approximate 8.4 kb genomic fragments including 6320-bp 5′ flanking sequence, the GSE5 gene and 1580-bp 3′ flanking sequence from 63 cultivated rice and 26 wild rice (O. rufipogon) were amplified and sequenced. The DNA sequences were aligned using the CLUSTAL X 2.1 program. The evolutionary history was inferred using the neighbour-joining method with the MEGA7.0 program.
The 488-bp sequence was amplified from the PCR products of crGSE5L-1 and crGSE5L-2 using the primers crGSE5L-1F and crGSE5L-2R and cloned into the vector pMDC99-Cas9 using in-fusion enzyme (Genebank Biosciences Inc, China) to generate the CRISPR/Cas9-GSE5L plasmid. The plasmids were introduced into Agrobaterium tumefaciens strain GV3101 by electroporation, and rice transformation was transformed according to a previous published method (Hiei et al., 1994).
Field-grown plants were raised during the standard rice season at Experimental Stations of the Institute of Genetics and Developmental Biology in Beijing. The spacing between plants was 20 cm.
Grain size of the Zhonghua 11 and GSE5-Like-crispr were measured using the SC Detection and Analysis System of Rice Seeds (Hangzhou WSeen Detection Technology). Actual yield of Zhonghua 11, GSE5-cr and proActin:GSE5 were weighted using electronic analytical balance (METTLER MOLEDO AL104 CHINA).
To evaluate the application potential of GSE5 for improving grain yield, we investigated yield traits of Zhonghua 11, GSE5-cr and proActin:GSE5 plants. Actual yield per plant in GSE5-cr was increased compared with that in Zhonghua 11 (
Oryza sativa GSE5 genomic sequence
Arabidopsis thaliana:
Glycine max:
Medicago truncatula:
Triticum aestivum:
Zea mays:
Oryza sativa:
Sorghum bicolor
Setaria italica
Setaria italica Primers for CRISPR/Cas9:
Z.mays
Sorghum bicolor:
MEDICAGO TRUNCATULA
Triticum aestivum
GLYCINE MAX
Arabidopsis
Arabidopsis:
Glycine max:
Medicago truncatula:
Sorghum bicolor:
Triticum aestivum:
Z.mays:
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/078137 | Mar 2017 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/050761 | 3/23/2018 | WO | 00 |
