Patent Application
20190085355
The invention relates to plants that are drought tolerant and related methods and uses.
Maize (Zea mays) production is frequently compromised by water scarcity, which is aggravated by trends of climate warming and erratic rainfall patterns on a global scale [1-2]. During the past two decades, although progress has been made towards an overall increase in maize yields, plant sensitivity to drought stress has increased [1]. It is proposed that to improve the ability of maize to overcome stress bottlenecks rather than primary productivity would be the primary driving force to produce higher yielding maize [3]. Thus, enhanced drought tolerance has become a priority trait in current maize genetic improvement efforts. However, the identification of the genetic components underlying drought tolerance has proven to be challenging. To date, no quantitative trait loci (QTLs) responsible for maize drought tolerance have been cloned, despite the reports of their mapping information [4-7].
Traditional QTL mapping, based on the genetic linkage of a certain trait with the molecular marker within a biparental segregation population, usually recombinant inbred lines (RILs), has been successfully used to identify the genes underlying QTLs for glume architecture [8], branched architecture [9], flowering time [10], photoperiod sensitivity [11], resistance to head smut [12] etc. However, it usually takes a relatively long time to generate RILs of the two parents, which are phenotypically contrasting with respect to the trait. Moreover, the mapping resolution is largely dependent on the genetic recombination among these RILs. To achieve an accurate mapping towards the gene cloning, it usually requires either a large number of RILs initially or several consecutive steps of selfing or backcrossing, in order to narrow down the region containing the candidate gene. Thus, a large number of QTL mapping studies have been conducted but only a limited number of them were cloned [13]. Another limitation of this approach is that the functional variation(s) of only two parental alleles can typically be evaluated during the mapping process.
Recently, genome-wide association study (GWAS), which is based on genetic linkage disequilibrium (LD) and makes use of natural variation and recombinants, has been used as a novel strategy for dissecting complex trait loci in plants [14-17]. Since the historical and revolutionary recombinant events can be exploited in a collection of a large number of genotypes, the LD mapping can reach a high resolution and investigate multiple alleles of a single locus [14]. With the development of high-throughput DNA variation discovery technology and improvement of statistical analyses, GWAS has gained favourability in genetic research in various plant species. Especially, due to the rapid LD decay in the maize genome, GWAS has facilitated the genetic dissection of several complex traits, including kernel β-carotene [18] and oil content [19], and flowering time in maize [20]. Although association studies of maize drought tolerance have been attempted [21-24], the proposed candidate genes or their causative variations still remain to be verified and resolved.
In the maize genome, ˜85% of the genomic contents are composed of transposable elements (TEs), and the generic sequences are embedded in a vast expanse of TEs [25]. In order to maintain stability of the genome, transposable elements (TEs) are usually silenced and inactive, due to DNA and chromatin modifications [26]. However, TEs have been shown to play important roles in plant evolution and environmental adaptation. For instance, a Hopscotch element inserted at ˜60-kb upstream of teosinte branched1 (tb1) increased maize apical dominance [9] and a CACTA-like transposable element located ˜2-kb upstream of ZmCCT was found to contribute to maize photoperiod sensitivity [20]. Miniature Inverted-repeat Transposable Elements (MITEs) are a kind of non-autonomous DNA transposon, which are usually shorter than 600 bp and widespread in plant genomes [27]. A MITE insertion in ˜70-kb upstream of ZmRAP2.7 was demonstrated to be associated with maize flowering time [10]. TEs can influence nearby gene expression either through the cis-acting element residing in their own sequences, or by changing the DNA or chromatin methylation status of adjacent genes [26, 28, 29]. In rice (Oryza sativa), MITEs have been recently discovered to be capable of generating 24-nt siRNA, depending on Dicer-like 3a (OsDCL3a) activity, and interfering with nearby gene expression through RNA-directed DNA methylation (RdDM) [30]. In plants, the RdDM pathway consists of the following major steps: (i) the RNA polymerase IV transcribes single-strand RNAs from repetitive heterochromatin regions, (ii) its physically associated RNA-dependent RNA polymerase 2 (RDR2) synthesizes the double-stranded RNA (dsRNAs), (iii) the dsRNAs are cleaved by Dicer-like 3 (DCL3) into 24-nt siRNAs, and (iv) ARGONAUTE 4 (AGO4) subsequently loads the siRNAs to their complementary DNA regions. Lastly, the formed complex recruits the DNA methyltransferase (DRM2) to catalyze methylation at cytosine in CG, CHG and CHH contents (H=A, G or C), especially in CHH sequence context, which is a hallmark of RdDM31-33. Enrichment of chromatin Histone3 lysine 9 dimethylation (H3K9me2), which is mainly catalyzed by a histone methyltransferase, SUVH4 (also named KYP), couples the DNA methylation in the adjacent regions [30, 34].
The inventors have shown by GWAS that an 82-bp (MITE) insertion in the promoter region of a NAC gene (ZmNAC111) is associated with maize drought tolerance. The MITE insertion correlates with lower ZmNac111 expression in maize, and when heterologously expressed in Arabidopsis it suppresses the ZmNac111 expression via the RdDM pathway. Transgenic studies demonstrated that enhanced expression of ZmNac111 conferred drought tolerance in both transgenic Arabidopsis and maize seedlings by improving plant water use efficiency (WUE) and enhancing the expression of stress-responsive genes under the stress. A comparison of MITE insertion frequency and nucleotide diversity at the ZmNac111 locus among teosinte, tropical/subtropical and temperate genotypes, suggests that the MITE insertion appears to have occurred after maize domestication from teosinte and spread in the temperate germplasm. The identification of this MITE insertion therefore provides an insight into the genetic natural variation of maize drought tolerance.
The inventors have identified and characterised the ZmNac111 promoter gene in maize and have surprisingly found that in strains where a miniature inverted-repeat transposable element (MITE) is inserted into the promoter this significantly affects drought tolerance. The inventors have also generated genetically altered, specifically, transgenic maize and Arabidopsis overexpressing ZmNac111, which displayed enhanced tolerance to drought stress compared to control plants that did not overexpress ZmNac111. These plants also did not show any growth penalties. The identification of ZmNac111 and its role in conferring drought tolerance is of significant value as this makes it possible to generate drought tolerant plants, which are important in agriculture.
The invention is thus aimed at providing genetically altered plants that show drought tolerance and related methods and uses. In one embodiment, the invention is aimed at providing transgenic plants that show drought tolerance and related methods and uses.
In a first aspect, the invention relates to a genetically altered plant or part thereof expressing a nucleic acid construct comprising a nucleic acid as defined in SEQ ID NO. 1, 2 or 3 or a functional homologue or variant thereof. In one embodiment, the invention relates to a transgenic plant or part thereof expressing a nucleic acid construct comprising a nucleic acid as defined in SEQ ID NO. 1, 2 or 3 or a functional homologue or variant thereof. The invention also relates to a product derived from a plant as defined above or from a part thereof.
In another aspect, the invention relates to a vector comprising a nucleic acid as defined in SEQ ID NO. 1, 2 or 3 or a functional homologue or variant thereof.
In another aspect, the invention relates to a host cell comprising a vector as described above.
In another aspect, the invention relates to a use of a nucleic acid as defined in SEQ ID NO. 1, 2 or 3 or a functional homologue or variant thereof or a vector as described above in conferring drought tolerance.
In another aspect, the invention relates to a use of a nucleic acid as defined in SEQ ID NO. 1, 2 or 3 or a functional homologue or variant thereof or a vector as described above in increasing yield/growth of a plant under drought stress conditions.
In another aspect, the invention relates to a method for increasing drought tolerance of a plant said method comprising introducing and expressing in said plant a nucleic acid construct comprising a nucleic acid as defined in SEQ ID NO. 1, 2 or 3 or a functional homologue or variant thereof.
In another aspect, the invention relates to a method for increasing yield of a plant under drought or water deficit conditions said method comprising introducing and expressing in said plant a nucleic acid construct comprising a nucleic acid as defined in SEQ ID NO. 1, 2 or 3 or a functional homologue or variant thereof.
In a further aspect, the invention relates to a method for producing a mutant plant tolerant to drought comprising introducing a mutation into the nucleic acid sequence of endogenous ZmNAC111 or the endogenous ZmNAC111 promoter or a functional homologue or variant thereof using targeted genome modification.
In a final aspect, the invention relates to a genetically altered plant, wherein said plant carries a mutation in the endogenous NAC111 gene or NAC111 promoter gene.
The invention is further described in the following non-limiting figures.
(maize
) inbred lines of maize. Nucleotide diversity was compared across the ZmNac111 locus, among the 72 MITE
and 190 MITE− maize inbred lines and 42 teosinte entries. ‘P’ denotes the ZmNac111 promoter region. Nucleotide diversity (it) for teosinte (itT, grey), the maize inbred lines of maize (it
, red) and Maize (it_, green) was calculated using a 100-bp sliding window and 25-bp step. The Tajima's D values of different regions are shown. *P<0.05; **P<0.01; ***P<0.001.
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. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or coding DNA. The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
As used herein, the term “genetically altered” includes, but is not limited to, transgenic plants and mutant plants.
For the purposes of the invention, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or
(b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
(c) a) and b)
are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above—becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815 both incorporated by reference.
The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, breeding methods, stable transformation methods, transient transformation methods, and virus-mediated methods. Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome.
A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the different embodiments of the invention are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. According to the invention, the transgene is stably integrated into the plant and the plant is preferably homozygous for the transgene. Thus, any off spring or harvestable material derived from said plant is also preferably homozygous for the transgene.
The aspects of the invention involve recombination DNA technology and in a preferred embodiment exclude embodiments that are solely based on generating plants by traditional breeding methods.
For the purposes of the invention, a “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 the mutagenesis methods described herein. In one embodiment, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, the endogenous ZmNac111 promoter sequences in wheat have been altered compared to wild type sequences using a mutagenesis method. These mutations may cause activation or otherwise enhance the activity of the ZmNac111 promoter or a functional homologue or variant thereof. Such plants have an altered phenotype and show tolerance or increased tolerance to drought compared to wild type plants. Therefore, the tolerance is conferred by the presence of a mutated endogenous ZmNac111 promoter gene in the wheat plant genome. In a preferred embodiment, the endogenous ZmNac111 promoter sequence is specifically targeted using targeted genome modification and is not conferred by the presence of transgenes expressed in wheat. In an alternative embodiment, there is provided a mutant plant that expresses a nucleic acid as defined in SEQ ID NO: 1, 2 or 3, or a functional homologue or variant thereof. Again, such plants have an altered phenotype and show tolerance or increased tolerance to drought compared to wild type plants. Also, again, in one embodiment, the phenotype of such plants is not conferred by the presence of a transgene(s).
The inventors have identified that a miniature inverted-repeat transposable element (MITE) that is inserted into the promoter of a NAC gene (ZmNAC111) significantly affects drought tolerance and that furthermore, overexpression of ZmNac111 in transgenic maize enhances drought tolerance.
A control plant as used herein is a plant which has not been modified according to the methods of the invention. Accordingly, the control plant has not been genetically modified or altered to express a nucleic acid as described herein. In one embodiment, the control plant is a wild type plant. In another embodiment, the control plant is a plant that does not carry a transgene according to the methods described herein, but expresses a different transgene. In another embodiment, the control plant is plant that has not been subjected to targeted genome modification or editing. The control plant is typically of the same plant species, preferably the same ecotype as the plant to be assessed.
Thus, in a first aspect, the invention relates to a genetically altered plant expressing a nucleic acid construct comprising a ZmNac111 nucleic acid sequence or a variant or homologue thereof. In one embodiment, the invention relates to a transgenic plant expressing a nucleic acid construct comprising a ZmNac111 nucleic acid sequence or a variant or homologue thereof Thus, the genetically altered, or in one embodiment, transgenic plant includes within its genome a nucleic acid construct comprising a ZmNac111 nucleic acid sequence. In one embodiment, wherein the plant is a transgenic plant, preferably, said plant is homozygous for the presence of the transgene.
In one embodiment, the ZmNac111 nucleic acid sequence comprises or consists of SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof. SEQ ID NO: 1 represents the genomic DNA. Residues 157 to 364, 534 to 872 and 968 to 1850 of SEQ ID NO: 1 are the coding regions (SEQ ID NO: 2). SEQ ID NO: 1 is the nucleotide sequence of ZmNac111 of the inbred maize line B73. The accession number is GRMZM2G127379. In a further embodiment, the ZmNac111 nucleic acid sequence comprises or consists of SEQ ID NO: 3 or a functional homologue or variant thereof. SEQ ID NO: 3 is the cDNA sequence of ZmNAC111.
The polypeptide encoded by SEQ ID NO: 1, 2, 3 or a functional homologue or variant thereof comprises or consists of SEQ ID NO: 4 or a functional homologue or variant thereof. Thus, the genetically altered, or, in one embodiment, transgenic plant of the invention expresses a ZmNac111 nucleic acid sequence and produces a protein that comprises or consists of SEQ ID NO: 4 or a functional homologue or variant thereof.
According to the various aspects of the invention, the term “functional homologue or variant of a nucleic acid sequence” as used herein, for example with reference to SEQ ID NO: 1, 2, 3 or 4 or homologs thereof, refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant ZmNac111 gene or ZmNac111 protein sequence, for example confers drought tolerance when expressed in a non-genetically altered or transgenic plant. A functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, 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, to the wild type sequences as shown herein and is biologically active.
Thus, it is understood, as those skilled in the art will appreciate, that the aspects of the invention, including the methods and uses, encompass not only a ZmNac111 nucleic acid or ZmNac111 protein sequence as described herein, for example a nucleic acid sequence comprising or consisting or SEQ ID NO: 1, 2, 3, a polypeptide comprising or consisting or SEQ ID NO: 4, but also functional homologues or variants of a ZmNac111 gene or ZmNac111 protein that do not affect the biological activity and function of the resulting protein. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do however 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. 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.
Generally, variants of ZmNac111 have at least 75% 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% overall sequence identity to the sequences represented by SEQ ID NO: 1, 2, 3 or 4.
A biologically active variant of a ZmNac111 protein may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. In certain embodiments, ZmNac111 proteins may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the ZmNac111 protein can be prepared by mutations in the DNA. 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. The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. When it is difficult, however, to predict the exact effect of a substitution, deletion, or insertion in advance of making such modifications, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
The term homologue as used herein also designates a NAC111 orthologue from other plant species. A homologue of ZmNac111 polypeptide has, 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 SEQ ID NO: 4. Preferably, overall sequence identity is more than 70% or more than 73%. Preferably, 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%. In another embodiment, the homologue of a NAC111 nucleic acid sequence has, 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 nucleic acid represented by SEQ ID NO: 1, 2 or 3. Preferably, overall sequence identity is more than 70% or more than 73%. Preferably, 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%. The overall sequence identity is determined using a global alignment algorithm known in the art, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys). Variants of homologs are also within the scope of the invention.
For example, sequence identity/similarity values provided herein can refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof.
As used herein, “sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid 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. When sequences differ in conservative substitutions, the percentage sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”.
In one embodiment of the plants, methods and uses described herein, the functional homologue is NAC10, for example OsNac10 (see SEQ ID NO: 5 and 6).
In one embodiment of the plants, methods and uses described herein, the functional homologue is as shown in SEQ ID No 5 or 6, 7 or 8, 9 or 10, 11 or 12, 13 or 14, 15 or 16 or a variant thereof.
In one embodiment, the ZmNAC111amino acid sequence encoded by SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof (SEQ ID NO: 4) is characterised by the presence of a conserved motif. In a functional ZmNac111 variant protein, changes to the amino acid sequence are preferably located outside these domains.
In one embodiment, a ZmNac111 homologue comprises a NAC DNA-binding domain. NAC DNA-binding domains are typically around 160 amino acids and are divided into five sub-domains, classed A, B, C, D and E and, in one embodiment, have the following consensus sequence:
or variants thereof. A variant is as defined herein.
In one embodiment, a NAC111 homologue comprises a NAC DNA-binding domain having the following sequence:
or a variant thereof. In one embodiment, the domain has at least 80%, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to this domain.
In a further embodiment, a ZmNac111 homologue comprises a NAC DNA-binding domain, as defined above (SEQ ID NO: 22) and/or is a functional homologue, meaning the homologue retains the biological function of the ZmNac111 gene or ZmNac111 protein sequence, for example confers drought tolerance when expressed in a genetically altered or, in one embodiment, transgenic plant and/or has, 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 SEQ ID NO: 4 or to the nucleic acid represented by SEQ ID NO: 1, 2 or 3.
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 when expressed in a plant. Thus, one of skill in the art will recognize that analogous amino acid substitutions listed above with reference to SEQ ID NO: 4 can be made in ZmNac111 from other plants by aligning the polypeptide sequence to be mutated with the ZmNac111 polypeptide sequence as set forth in SEQ ID NO: 4.
Thus, the nucleotide sequences of the invention and described herein can 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. 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.
Preferred homologues of ZmNac111 peptides are ZmNac111 homologues from crop plants, for example cereal crops. In one embodiment, preferred homologues include maize, rice, wheat, sorghum, sugar cane, oilseed rape (canola), soybean, cotton, potato, tomato, tobacco, grape, barley, pea, bean, field bean or other legumes, lettuce, sunflower, alfalfa, sugar beet, broccoli or other vegetable brassicas or poplar. Preferred homologues and their peptide sequences are also shown in SEQ ID Nos 5 to 16.
Also, the various aspects of the invention the aspects of the invention, including the methods and uses, encompass not only a ZmNac111 nucleic acid sequence as shown herein, but also a fragment thereof. By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence confer drought tolerance.
In one embodiment according to the various aspects of the invention, the nucleic acid construct comprises a regulatory sequence or element. According to the various aspects of the invention, the term “regulatory element” is used interchangeably herein with “control sequence” and “promoter” and all terms are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term “regulatory element” also includes terminator sequences which may be included 3′ of the ZmNac111 nucleic acid sequence. The term “promoter” typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences.
The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
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. For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes are known to the skilled person and include for example beta-glucuronidase or beta-galactosidase.
In one embodiment, the ZmNac111 nucleic acid is operably linked to a regulatory sequence or element. 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 preferred embodiment, the nucleic acid sequence may be expressed using a promoter that drives overexpression. Overexpression according to the invention means that the transgene is expressed at a level that is higher than expression of endogenous counterparts driven by their endogenous promoters. For example, overexpression may be carried out using a strong promoter, such as 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 the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, rubisco small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that gives enhanced expression. Alternatively, enhanced or increased expression can be achieved by using transcription or translation enhancers or activators and may incorporate enhancers into the gene to further increase expression. Furthermore, an inducible expression system may be used, where expression is driven by a promoter induced by environmental stress conditions, in particular drought. The promoter may also be tissue-specific. The types of promoters listed above are described in the art. Other suitable promoters and inducible systems are also known to the skilled person.
In a one embodiment, the promoter is a constitutive or strong promoter. In one embodiment, the promoter is Zmubi1. In one embodiment, the promoter has the sequence of SEQ ID NO: 23, or a variant as defined herein.
In another embodiment the regulatory sequence is the CaMV35S promoter.
In one embodiment, the promoter is a ZmNac111 promoter isolated from a drought tolerant maize inbred line. Such promoter does not contain a polymorphism at the following position: InDel-572 with respect to SEQ ID NO: 2 (the A in the ATG site is designated as +1; this is the first residue in SEQ ID NO: 2) compared to a drought sensitive line. In particular, this promoter can be used to express ZmNac111 at the onset of drought stress. In one embodiment, the ZmNac111 promoter or variant thereof, has the sequence SEQ ID NO: 24
Additional nucleic acid sequences which facilitate cloning of the target nucleic acid sequences into an expression vector may also be included in the nucleic acid construct according to the various aspects of the invention. This encompasses the alteration of certain codons to introduce specific restriction sites that facilitate cloning. A terminator sequence may also be included in the construct.
In one embodiment, the plant is maize and the nucleic acid construct comprising ZmNac111 may be expressed in a maize plant by recombinant methods. In another embodiment, an exogenous ZmNac111 nucleic acid is expressed in a second plant of another species by recombinant methods. Thus, all aspects of the invention, including the genetically altered, e.g. transgenic plants and methods of the invention, also extend to plants other than maize which express a nucleic acid construct comprising a ZmNac111 nucleic acid sequence.
In one embodiment, the plant is a monocot or dicot plant. In one embodiment, the plant is a crop plant or biofuel plant.
In one embodiment of the various aspects of the invention, the plant is a dicot plant. A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species. In one embodiment, the plant is oilseed rape.
Also included are biofuel and bioenergy crops such as rape/canola, corn, sugar cane, palm trees, jatropha, soybeans, sorghum, sunflowers, cottonseed, Panicum virgatum (switchgrass), linseed, wheat, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).
In one embodiment of the various aspects of the invention, the plant is a dicot plant. A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana.
In preferred embodiments of the various aspects of the invention 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 preferred embodiments of the various aspects of the invention the plant is selected from a grain plant, an oil-seed plant, and a leguminous plant.
Most preferred plants according to the various aspects of the invention are maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
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, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. 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 gene/nucleic acid of interest.
The term “maize” as used herein refers to a plant of the Zea mays L. ssp. mays and is also known as “corn”. The term “maize plant” includes: whole maize plants, maize germplasm, maize plant cells, maize plant protoplast, maize plant cell or maize tissue cultures from which maize plants can be regenerated, maize plant calli, and maize plant cells that are intact in maize plants or parts of maize plants, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips, and the like. The maize can be an inbred line, or a maize hybrid such as a maize single cross hybrid.
The various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
The invention also extends to harvestable parts of a plant of the invention as described above such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The invention furthermore relates 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. The invention also relates to products, including food products and food supplements comprising the plant of the invention or parts thereof.
The plant according to the invention shows increased resistance or tolerance to drought or water deficiency compared to a control plant.
In one embodiment, said stress is moderate or severe stress. A plant according to the invention also shows reduced growth/yield penalties under moderate stress compared to a control plant.
In one embodiment, the methods of the invention thus relate to increasing resistance or tolerance to moderate (non-lethal) stress or severe stress. In the former embodiment, genetically altered, or in one embodiment, transgenic plants according to the invention show increased resistance or tolerance to stress and therefore, the plant yield is not or less affected by the stress compared to wild type yields which are reduced upon exposure to stress. In other words, an improvement in yield under moderate stress conditions can be observed.
For example, drought tolerance is assessed predominantly under quite severe conditions in which plant survival is scored after a prolonged period of soil drying. However, in temperate climates, limited water availability rarely causes plant death, but restricts biomass and seed yield. Moderate water stress, that is suboptimal availability of water for growth can occur during intermittent intervals of days or weeks between irrigation events and may limit leaf growth, light interception, photosynthesis and hence yield potential. Leaf growth inhibition by water stress is particularly undesirable during early establishment. There is a need for methods for making plants with increased yield under moderate stress conditions. In other words, whilst plant research in making stress tolerant plants is often directed at identifying plants that show increased stress tolerance under severe conditions that will lead to death of a wild type plant, these plants do not perform well under moderate stress conditions and often show growth reduction which leads to unnecessary yield loss. Thus, in one embodiment of the methods of the invention, yield is improved under moderate stress conditions. The genetically altered, such as transgenic plants according to the various aspects of the invention show enhanced tolerance to these types of stresses compared to a control plant and are able to mitigate any loss in yield/growth. The tolerance can therefore be measured as an increase in yield as shown in the examples. The terms moderate or mild stress/stress conditions are used interchangeably and refer to non-severe stress. In other words, moderate stress, unlike severe stress, does not lead to plant death. Under moderate, that is non-lethal, stress conditions, wild type plants are able to survive, but show a decrease in growth and seed production and prolonged moderate stress can also result in developmental arrest. The decrease can be at least 5%-50% or more. Tolerance to severe stress is measured as a percentage of survival, whereas moderate stress does not affect survival, but growth rates. The precise conditions that define moderate stress vary from plant to plant and also between climate zones, but ultimately, these moderate conditions do not cause the plant to die. Importantly, the Inventors have identified that there are no growth penalties observed in the genetically altered plants described herein.
Generally speaking, moderate drought stress is defined by a water potential of between −1 and −2 Mpa.
In one embodiment, the maize relative leaf water content (RLWC) at 95-100% is well-watered or favourable growth condition; RLWC at around 70-65% is moderate drought stress; RLWC at around 58-55% is severe drought stress.
Drought tolerance can be measured using methods known in the art, for example assessing survival of the genetically altered plant compared to a control plant, through leaf water potentials or by determining turgor pressure, rosette radius, water loss in leaves, growth or yield. Drought tolerance can also be measured by assessing stomatal conductance (Gst) and transpiration in whole plants under basal conditions.
According to the invention, a genetically altered plant, such as, in one embodiment, a transgenic plant has enhanced drought tolerance if the survival rates are at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold higher than those of the control plant after exposure to drought and/or after exposure to drought and re-watering. Also according to the invention, a genetically altered plant, such as a transgenic plant has enhanced drought tolerance if the rosette radius is at least 10, 20, 30, 40, 50% larger than that of the control plant after exposure to drought and/or after exposure to drought and re-watering. The plant may be deprived of water for 10-30, for example 20 days and then re-watered. Also according to the invention, a genetically altered plant, such as a transgenic plant has enhanced drought tolerance if stomatal conductance (Gst) and transpiration are lower than in the control plant, for example at least 10, 20, 30, 40, 50% lower.
The terms “increase”, “improve” or “enhance” are interchangeable. Yield for example is increased by at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40% or 50% or more in comparison to a control 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, or 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. The term “yield” of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant. Thus, according to the invention, yield comprises one or more of and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased number of seed capsules/pods, increased seed size, increased growth or increased branching, for example inflorescences with more branches. Preferably, yield comprises an increased number of seed capsules/pods and/or increased branching. Yield is increased relative to control plants.
In another aspect, the invention relates to an isolated nucleic acid comprising or consisting of SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof. In another aspect, the invention relates to an isolated amino acid sequence comprising or consisting of SEQ ID NO: 4 or a functional homologue or variant thereof.
In another aspect, the invention relates to a vector comprising a nucleic acid construct comprising SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof. In one embodiment, said vector is an expression vector. Expression vectors for expressing nucleic acid sequences in a plant are well known. An example is pGXX. For example, a ZmNac111 nucleic acid sequence as described herein can be inserted between the Smal and Sall restriction sites of the pGXX vector. Plant expression vectors also include dual agrobacterium vectors and plant micro bombardment vectors such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pSBII, pCAMBIA1391-Xa or pCAMBIA1391-Xb.
The vector may further comprise a regulatory sequence which directs expression of the nucleic acid. Such sequences are described elsewhere herein. In one example, the regulatory sequence is a promoter that directs overexpression of the nucleic acid sequence. Marker genes (e.g Gus) and resistance genes can also be included.
In another aspect, the invention relates to a host cell comprising a vector as described herein. The host cell can be selected from a plant cell or a bacterial cell, for example Agrobacterium. The invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described above.
In another aspect, the invention relates to the use of a nucleic acid construct comprising or consisting of SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof or a vector described herein in conferring drought tolerance to a plant.
In another aspect, the invention relates to the use of a nucleic acid construct comprising or consisting SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof or a vector described herein in increasing yield/growth of a plant under drought stress conditions.
In another aspect, the invention relates to a method for conferring to or increasing drought tolerance of a plant said method comprising introducing and expressing in said plant a nucleic acid construct comprising or consisting of SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof. In another aspect, the invention relates to a method for increasing yield of a plant, for example under moderate drought stress, said method comprising introducing and expressing in said plant a nucleic acid construct comprising or consisting of SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof.
The term plant is defined elsewhere herein.
In one embodiment, said construct further comprises a regulatory sequence. Such sequences are described elsewhere herein. In one example, the regulatory sequence is a promoter that directs overexpression of the nucleic acid sequence.
The nucleic acid or vector described above is used to generate genetically altered, and in one embodiment, transgenic plants using transformation methods known in the art. Thus, according to the various aspects of the invention, a nucleic acid comprising a ZmNac111 nucleic acid or a functional homologue or variant thereof is introduced into a plant and expressed as a transgene. The nucleic acid sequence is introduced into said plant through a process called transformation. The term “introduction” or “transformation” as referred to herein encompass 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 polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. 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. Advantageously, any of several transformation methods may be used to introduce the gene 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, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.
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. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using 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. 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).
The invention relates to a method for producing a genetically altered plant with improved drought tolerance compared to a control plant comprising
Thus, the invention relates to a method for producing a genetically altered plant with improved yield under water deficiency or drought stress comprising
In one embodiment, the drought stress is moderate.
The methods above may comprise the further steps of:
The invention also relates to plants obtained or obtainable with said method. The term plant is defined elsewhere herein.
The invention also relates to a plant with increased expression of an endogenous nucleic acid as defined in SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof wherein said endogenous promoter (SEQ ID NO: 24) carries a mutation introduced by mutagenesis or genome editing which results in increased expression of the nucleic acid as defined in SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof.
The invention also relates to a method for increasing expression of a nucleic acid as defined in SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof in a plant, producing plants, a method for mitigating the impacts of stress conditions on plant growth and yield and a method for producing plants with improved yield/growth under stress conditions comprising the steps of mutagenising a plant population, identifying and selecting plants with an improved yield/growth under stress conditions and identifying a variant ZmNac111 promoter sequence which directs expression of a nucleic acid as defined in SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof. The above can be achieved using targeted genome editing.
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 customizable 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 its 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. Reference 71 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.
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.
Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and 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: CRIPSR 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.
Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.
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
Accordingly in one aspect of the invention there is provided a method for producing a mutant plant resistant to drought comprising introducing a mutation into the nucleic acid sequence of the endogenous ZmNac111 promoter or a functional homologue or variant thereof using targeted genome modification. In a preferred embodiment, the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9.
In one embodiment, the ZmNac111 promoter or a functional homologue or variant thereof is isolated from a drought-resistant maize inbred line. In an alternative embodiment, the ZmNac111 promoter or a functional homologue or variant thereof is isolated from a drought-sensitive maize inbred line.
In one embodiment, the ZmNac111 promoter is represented by SEQ ID NO: 24
In an alternative embodiment, the ZmNac111 promoter is represented by SEQ ID NO: 25 (this sequence includes the MITE).
In one embodiment targeted genome editing or modification as defined above is used to delete at least one residue from the nucleic acid sequence of the ZmNac111 promoter, or a functional homologue or variant thereof. In a preferred embodiment, the targeted genome editing is used to delete the following sequence from a ZmNac111 promoter containing the below sequence (for example, SEQ ID NO: 26), or a functional homologue or variant thereof
In another embodiment, targeted genome editing is used to insert at least one nucleic acid in the nucleic acid sequence of the ZmNac111 promoter, or a functional homologue or variant thereof. In a preferred embodiment, this mutation enhances activity of the endogenous promoter. For example, targeted genome modification can be used to insert at least one enhancer or promoter site, such as a TATA box (TATAAA), a GC box (GGGCGG) or a CAAT (GGCCAATCT) box, or functional variants thereof.
The invention also relates to plants obtained or obtainable with said method. The term plant is defined elsewhere herein. There is also provided the use of a mutated endogenous ZmNac111 promoter as described above or a functional homologue or variant thereof to increase yield and/or growth of a plant under drought stress conditions. Alternatively, there is also provided the use of a mutated endogenous ZmNac111 promoter as described above or a functional homologue or variant thereof to confer drought tolerance.
In a final aspect of the invention there is provided a genetically altered plant expressing a nucleic acid construct comprising a nucleic acid as defined in SEQ ID NO: 1, 2 or 3 or a functional homologue or variant thereof, wherein the nucleic acid further comprises at least one of the following mutations: SNP1532: C/A and/or SNP1535:A/G. In one embodiment, the plant is a mutant plant. There is also provided a method for producing a genetically altered plant resistant to drought comprising introducing a mutation into the nucleic acid sequence of SEQ ID NO: 1, 2 or 3, wherein said mutation is SNP1532:C/A and/or SNP1535:A/G. Again, in a preferred embodiment, the plant is a mutant plant.
In all aspects of the invention where we discuss conferring drought tolerance, the invention can equally apply to conferring drought resistance.
A further objective of the present invention is to provide a plant drought tolerant related protein ZmNAC111, an encoding gene thereof and an application thereof.
The protein provided in this invention is derived from corn (Zea mays L.) and has a name of ZmNAC111. Said protein is a protein of a) or b):
a) a protein consisting of an amino acid sequence shown in SEQ ID NO. 27 in the sequence list;
b) a protein derived from the protein a), wherein one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 27 is substituted, removed and/or added, and the protein relates to drought tolerance.
The amino acid shown in SEQ ID NO. 27 consists of 475 amino acid residues. In order to make the protein in (a) easier for purification, a tag shown in Table 1 can be applied to connect to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO. 27.
The protein in (b) can be obtained by artificial synthesis or obtained by synthesizing the encoding gene first and then expressing the gene biologically. The encoding gene of protein (b) can be obtained by deleting one or more codons of amino acid residues, carrying out a missense mutation of one or more bases, and/or adding a tag as shown in Table 1 to the 5′ terminal and/or 3′ terminal, of the DNA sequence shown by locus 157-1584 of SEQ ID No.28.
Encoding the above-mentioned DNA molecule of the protein also falls into the protection scope of the invention.
Said DNA molecule is a DNA molecule of 1), 2), 3) or 4):
SEQ ID No. 28 consists of 1824 deoxynucleotides and it is the whole length cDNA sequence encoding said protein, wherein locus 157-1584 is the coding region.
The above-mentioned strict condition can be as follows: performing hybridization in a mixed solution of 7% lauryl sodium sulfate (SDS), 0.5M Na3PO4 and 1 mM EDTA at 50° C., and washing in 2×SSC and 0.1% SDS at 50° C.; or performing hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1 mM EDTA at 50° C., and washing in 1×SSC and 0.1% SDS at 50° C.; or performing hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1 mM EDTA at 50° C., and washing in 0.5×SSC and 0.1% SDS at 50° C.; or performing hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1 mM EDTA at 50° C., and washing in 0.1×SSC and 0.1% SDS at 50° C.; or performing hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1 mM EDTA at 50° C., and washing in 0.1×SSC and 0.1% SDS at 65° C.; or performing hybridization in a mixed solution of 6×SSC, 0.5% SDS at 65° C. and washing the membrane in 2×SSC and 0.1% SDS and 1% SDS respectively.
Recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses containing the above-mentioned DNA molecules also fall into the protection scope of the present invention.
Recombinant expression vectors containing said genes can be constructed with the existing plant expression vectors. Said plant expression vectors contain dual agrobacterium vectors and vectors that can be used for plant bombardment and so on. Examples are pROKII, pBin438, pCAMBIA1302, pCAMBIA 2301, pCAMBIA 1301, pCAMBIA 1300, pBI121, pCAMBIA 1391-Xa or pCAMBIA 1391-Xb (CAMBIA Cor.) and so on. Said plant expression vectors may also contain non-translational domains in the 3′ terminal of foreign genes, namely contain a polyadenylation signal or any other DNA fragments involving in the process of mRNA modification or gene expression. Said polyadenylation signal can direct a polyadenylic acid into the 3′ terminal of an mRNA precursor, for example non-translational domains of 3′ terminal transcriptions such as agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase Nos genes), and plant genes (such as soybean storage protein genes) all possess the similar functions.
When using said genes to construct the recombinant plant expression vectors, any kinds of enhancing type of promoters (such as cauliflower mosaic virus (CAMV) 35S promoters, corn ubiquitin promoters (Ubiquitin)), constitutive promoters or tissue specific expression promoters (such as seed specific expression promoters) can be added before the initial nucleotide transcription, all of which can be used separately or in combination with other promoters. Besides, when using the genes in this application to construct the plant expression vectors, an enhancer including a translational enhancer or a transcription enhancer can also be used. These enhancer domains can be ATG initial promoters or initial promoters in the adjacent domain, but they must be the same with the reading frame of the encoding sequences so as to ensure the correct translation of the entire sequence. Said translational control signals and initial promoters have a wide source and they can be obtained from natural source or can be artificially synthesized. The translational initial domain can originate from the transcription domain or structural genes.
In order to identify or select the transgenic plant cells or plants, plant expression vectors can be modified, for example, by adding genes that can express in plants (GUS gene, luciferase gens and so on) and encode enzymes capable of producing colour changes or luminous compounds; tag genes for antibiotics (such as nptll genes that give resistance to kanamycin and related antibiotics, bar genes that give resistance to herbicide phosphinothricin, hph genes that give resistance to antibiotic hygromycin and dhfr genes that give resistance to methatrexate as well as EPSPS genes that give resistance to glyphosates) or tag genes for anti-chemical agents (such as anti-herbicide genes); as well as mannose-phoshpate isomerase genes providing the metabolic capability for mannose.
In one embodiment, the recombinant vectors are pGZ or pSBIII.
The recombinant vector pGZ is the DNA fragment (downstream of 35S promoter) between Not I and Xho I cleavage sites of ZmNAC111 replacement vector pGKX shown by locus 157-1584 in SEQ ID NO. 28, while other sequences on the vector remain unchanged.
The recombinant vector pSBIII is the DNA fragment (downstream of Zmubil promoter) between Sma I and Hind III cleavge sites of ZmNAC111 replacement vector pSB II shown by locus 157-1584 in SEQ ID NO. 28, while other sequences on the vector remain unchanged.
The use of the above-mentioned proteins, DNA molecules or recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses in modulating the adversity resistance of a plant is also within the ambit of the present invention.
In said use, modulating the stress resistance of a plant is to improve the stress resistance of the plant, and said stress resistance is drought tolerance. In one embodiment, the plant is a monocotyledon or dicotyledon.
The use of the above-mentioned proteins, DNA molecules or recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses in cultivating a transgenic plant having stress resistance is also within the ambit of the present invention. Again, preferably, said stress resistance is drought tolerance, and preferably, said plant is a monocotyledon or dicotyledon.
Another objective of the present invention is to provide a method of cultivating a transgenic plant having a stress resistance, comprising the step of: introducing the above-described DNA molecule into a target plant to obtain a transgenic plant; the transgenic plant has a higher stress resistance than the target plant. In the above method, the stress resistance is drought tolerance and said plant is a monocotyledon or dicotyledon.
In the above method, said DNA molecule is introduced into the target plant through a recombinant vector pGZ or pSBIII.
Preferably, in the above method or use, said dicotyledon can specifically be Arabidopsis thaliana and said monocotyledon can specifically be corn (Zea mays L.).
The drought tolerance can be expressed with the following properties:
Accordingly, the invention can be described in the following aspects:
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.
All documents mentioned in this specification, including reference to sequence database identifiers, are incorporated herein by reference in their entirety. Unless otherwise specified, when reference to sequence database identifiers is made, the version number is 1.
“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 invention is further described in the following non-limiting examples.
To identify genes associated with maize drought tolerance, we performed GWAS by analyzing a natural maize population, consisting of 368 inbred lines that were collected from tropical/subtropical (TST) and temperate regions of the world [19]. Approximately, 560,000 single nucleotide polymorphisms (SNPs)[19] were applied to the study. Considering the complexity of plant drought tolerance, which is affected by both the time period and intensity of the stress imposed to plants, we decided to focus on tolerance to severe drought stress at the seedling stage. Seedling survival rate (SR) can reflect plant tolerance mechanisms and cellular responses to drought. It is less affected by environmental fluctuation, which helps to identify the underlying genetic determinant(s). The drought tolerance of each genotype was assayed by calculating a SR index (percentage of the survived plants after re-watering) under severe drought stress at seedling stage [24]. On average, the inbred lines from TST exhibited higher SR in comparison with those from temperate regions, indicating that maize germplasm derived from areas near to the place of origin may be more drought tolerant than those cultivated in temperate regions. On the whole genome scale, in consideration of the population structure (Q) and parental relatedness (K) of the population, a SNP within GRMZM2G127379 on chromosome 10 was identified to be significantly associated with plant drought tolerance (
NAC proteins regulate multiple biological processes in plants, including cotyledon [37] and root development [38], formation of secondary walls [39], leaf senescence [40], nutrient remobilization to grains [41], and stress responses [42]. Considering the possible function of NAC-type genes in plant drought tolerance, we then sequenced the ZmNac111 gene in 262 maize inbred lines. A 2.3-kb genomic region, spanning the 5′ to 3′-untranslation region (UTR) of ZmNAC111, was analyzed. A total of 157 SNPs and 119 InDels (Insertions and Deletions) were further identified. A newly identified 82-bp InDel (InDel-572), located 572-bp upstream of the start codon of ZmNAC111, was found to have the greatest significant association with the seedling SR (P=5.52×10-6, calculated under mixed linear model, see Methods,
Sequence analysis of InDel-572 in the promoter region of ZmNac111 gene revealed that it is composed of a long terminal inverted repeat (TIR) (38-bp for each), a 4-bp loop and two additional nucleotides “TA” at the end. Another “TA” sequence was found directly prior to this insertion (
Since the MITE insertion locates in the promoter region of ZmNAC111, we hypothesized that it results in altered ZmNac111 expression among different genotypes. To examine this hypothesis, we analyzed the expression of the ZmNac111 gene among 133 inbred lines under well-watered, moderate, and severe drought conditions. Quantitative RT-PCR (qRT-PCR) analysis of a total of 399 RNA samples revealed that ZmNac111 expression positively correlated with plant survival rate under both moderate and severe drought stresses, but not under well-watered conditions (
To further test whether the MITE insertion is the causative variant at the ZmNac111 locus, we constructed three bi-parental F2:3 populations. The genotypes of two parents of each population were either MITE+ or MITE− at the ZmNac111 locus. We observed that the MITE− allele co-segregated with drought tolerance in three bi-parental F2:3 populations (
To understand how the 82-bp MITE reduces ZmNac111 expression, we then checked the DNA and histone methylation status of ZmNac111 in the MITE− and MITE+ genotypes. Eight regions (R1-R8) spanning the ZmNac111 gene and promoter in two inbred lines, B73 (drought-sensitive, MITE+) and CIMBL55 (drought-tolerant, MITE−), were representatively analyzed. Results revealed that only R1 and R2, nearest the MITE insertion, were hypermethylated in ZmNAC111-B73 but not in ZmNAC111-CIMBL55, regardless of the stress treatment (
A considerable number of 21-nt to 24-nt small interference RNA (siRNA) species were identified that could be aligned to the MITE sequence in the maize small RNA database (http://sundarlab.ucdavis.edu/smrnas/) (
In order to verify that the MITE insertion mediated ZmNac111 repression through the RdDM pathway, five Arabidopsis mutants defective in key genes involved in the RdDM pathway were crossed with two independent homozygous 35S:gZmNAC111-B73-2 and -23 lines to transfer the 35S:gZmNAC111-B73 construct to the RdDM-defective backgrounds. In the drm1-2;drm2-2, ago4-5, and rdr2-2 mutants, ZmNAC111-B73 expression was greatly enhanced, while its DNA methylation and H3K9me2 were reduced to a comparable level relative to those of the ZmNAC111-CIMBL55 in the wild-type background. In the suvh4-3 mutant, the H3K9me2 level of ZmNAC111-B73 was significantly reduced, but the DNA hypermethylation remained relatively similar to that in the wild-type background, which was consistent with the fact that the SUVH4 functions as a histone methytransferase. In dcl2-1;dcl4-2, the gene expression, DNA methylation and H3K9me2 levels of ZmNAC111-B73 still differed from those observed for the ZmNAC111-CIMBL55 in wild-type background; most likely due to the intact DCL3 function in this mutant (
Given that ZmNac111 expression is positively correlated with maize drought tolerance, we generated both transgenic Arabidopsis and maize, overexpressing the coding sequence of ZmNac111 (from B73 genotype). For the transgenic Arabidopsis, the phenotypes of three independent 35S:ZmNAC111 lines were analyzed. In comparison with the empty-vector transformed plants (VC), the transgenic Arabidopsis displayed significantly enhanced drought tolerance, without remarkable morphological changes under normal growth conditions. When the survival of VC was around 20%, approximately 80% of the transgenic plants were alive in the parallel water-withholding experiments (
Similar improved drought tolerance was also observed in pot-grown transgenic maize transformed by ZmUbi:ZmNAC111. Under drought stress, approximately 80% of the T2 generation transgenic maize plants survived; whereas, the survival rate of the transgenic-negative sibling plants (WT) was only 30% (
In the next line of our study, we compared the transcriptome of the transgenic maize and WT plants under favourable and drought conditions. A total of 628 and 443 genes were found to be up- and down-regulated by two-fold in the transgenics compared with WT, respectively, under well-watered conditions (
Teosinte (Zea mays ssp.), a type of wild Mexican grass, is recognized as the direct progenitor of maize, based upon the fact that the natural cross of teosinte and maize are fertile and the availability of a wealth of genetic domestication information8,9. After domestication from teosinte, maize cultivation spread from TST to temperate regions and maize became a major crop plant providing nutritional calories for consumption by human beings. We were interested to know the presence and distribution of the 82-bp MITE insertion in the teosinte ZmNac111 locus. When 96 teosinte accessions were genotyped, none of them were found to carry the MITE insertion at ZmNAC111, indicating that this insertion might have occurred after the domestication of maize. Moreover, among 116 TST inbred lines, 10.34% of them were MITE+. Whereas, among 146 temperate lines (including stiff-stalk (SS), non-stiff-stalk (NSS), and Mixed origins51), 41.78% were MITE+(
The results are shown in
The above results show that protein ZmNAC111 and its encoding genes possess the function of regulating the drought tolerance of a plant. The overexpression of the encoding gene of the protein ZmNAC111 in plants can improve the drought tolerance of plants.
In this research, we reported that the natural variation of ZmNac111 gene is associated with maize drought tolerance on the whole genome-scale. By using a combined approach of GWAS and transgenic studies, we demonstrated that the causative variation at the ZmNac111 locus is likely an 82-bp MITE insertion in the gene promoter, which represses the gene expression through DNA and histone hypermethylation via the RdDM pathway. Our findings highlight the likely regulatory function of a TE in maize stress response and provide important insights into the genetic basis of the natural variation in maize drought tolerance; as well as new genetic strategies for improving this trait.
We identified the 82-bp MITE insertion in a location that was 572-bp upstream of the ZmNac111 coding region which was correlated with lower ZmNac111 expression and drought susceptibility (
TFs play important roles in the regulation of gene expression in response to abiotic stresses, and their molecular engineering is proposed as a potential strategy for the genetic improvement of stress tolerance in crops [54,55]. NAC proteins constitute a plant-specific superfamily whose members participate in various regulatory and developmental processes, including stress response and tolerance [42]. The typical NAC proteins share a conserved N-terminal DNA-domain, but vary greatly in other regions; resulting in distinct functions of different proteins. In maize, at least 116 predicted NAC members have been identified [35]. Although ZmNac111 was classified into an identical phylogenetic clade with OsNAC10 among the annotated NAC proteins examined (
It is considered that enhancing effective use of water (EUVV), which implies maximal soil moisture capture, reduced non-stomatal water loss and management for minimal soil evaporation, is important for drought tolerance improvement under field conditions; however, improving WUE by reducing SC and TR may diminish plant yield in fields [56]. In this research, we did not observe obvious morphological changes in transgenic plant overexpressing ZmNac111 in comparison with the control plants, when they grew in pots under favorable greenhouse conditions (
In addition to SNPs, TE presence/absence variations are common and widely distributed in the maize genome, which is considered a driving force for crop evolution and domestication [57]. The MITE insertion was only present in maize germplasm but not in the teosinte accessions we examined (
Methods
ZmNAC111-Gene Association Mapping.
GWAS of maize drought-tolerant genes was performed by analyzing a maize natural variation panel consisting of 368 inbred lines collected from TST and temperate regions19. Plant drought tolerance of different inbred lines was phenotypied as previously described24. Briefly, the natural variation panel of maize consisting of 368 maize inbred lines was planted in a cultivation pool (6×1.4×0.22 m, length×width×depth) in which 5-ton of loam were mixed with 0.25-ton of chicken manure. To phenotype the drought tolerance of each genotype, watering was withheld when the seedlings developed three true leaves. Re-watering was applied to recover the surviving plants when clear wilting difference was observed. After rehydration for 6 days, the survival rate of each genotype was scored. The phenotypic data were obtained from 6 replicated experiments. The 56,110 genomic and 525,105 transcriptomic SNPs, with minor allele frequency (MAF)≥0.05, were used for GWAS. The standard mixed linear model was applied (TASSEL 3.1.0)59, in which the population structure (Q) and kinship (K) were estimated as previously described24. Briefly, principle components of the association panel were calculated by EIGENSTRAT60 using the high-quality 52,5105 SNP data with MAF 0.0561. The first two dimensions were used in the principle component analysis (PCA) to estimate the population structure, which could explain the 11.01% of the phenotypic variation. These results were comparable to those that were calculated by STRUCTURE. The analysis was completed by the Im function in an R program. Single-marker association analysis was initially performed to filter out markers that had no relationship with the trait (p 0.995). Subsequently, 1,822 SNP markers on each chromosome were chosen to estimate the kinship coefficient (K) by SPAGeDi. Markers that were in approximate linkage equilibrium with each other were determined from PLINK62 based on SNP pruning (window size 50, step size 50, the LD R2 threshold is 0.2) and the number of the subset markers was 85,806. The suggestive P-value threshold to control the genome-wide type 1 error rate was 1.17×10-5, which was considered as the significance cutoff for the association. ZmNAC111-based association mapping was performed within 146 temperate and 116 TST maize inbred lines, which were representative of the whole population. The ZmNac111 promoter (˜0.7 kb), coding regions (include introns), and 5′ and 3′-UTR sequences were amplified and sequenced. These sequences were assembled using ContigExpress in Vector NTI Advance 10 (Invitrogen) and aligned using MEGA version 5 (http://megasoftware.net/). Polymorphisms (SNPs and InDels) were identified and their association to drought tolerance was calculated again by TASSEL 3.1.0, under the standard MLM, with MAF 0.05.
ZmNAC111 Gene Expression Analysis in Different Inbred Lines.
ZmNAC111 expression was analyzed in 133 maize inbred lines. Drought treatment was applied to the soil-grown plants at the 3-leaf seedling stage by withholding water. Leaf samples were collected when the relative leaf water content (RLWC) decreased from approximately 98% to 70%, and to 58%. Total RNA of 399 samples was isolated using TRIZOL reagent (Biotopped) from a minimum of 3 seedlings. RNA was treated with RNase-free DNasel (Takara), and single-stranded cDNA was synthesized using Recombinant M-MLV reverse transcriptase (Promega). The quantification method (2−ΔCt)63 was used and the variation in expression was estimated using three biological replicates. The maize Ubi-2 (UniProtKB/TrEMBL; ACC: Q42415) gene was used as an internal control to normalize the data. PCR conditions consisted of an initial denaturation step at 95° C. for 10 min, followed by 40 cycles at 95° C. for 15 sec, 60° C. f0 sec.
Allelic Effect of ZmNac111 in Maize Segregating Populations.
Three F2:3 segregating populations (CIMBL91×BY4944, CIMBL55×GEMS54 and CIMBL55×CIMBL9) were constructed. The genotype at the ZmNac111 locus was analyzed in approximately 200 individual F2 plants in each population. Polymorphisms in the PCR products were visualized on 2% agarose gels. Homozygous F2 individuals at the ZmNac111 locus of the tolerant allele (MITE−) and the sensitive allele (MITE+) were self-pollinated to obtain F3 progenies. F3 progenies that were homozygous at the InDel-572 locus (MITE−/− or MITE+/+) were mixed, respectively. Two types of F3 plants were grown in enriched soil (soil to vermiculite in a ratio of 1:1) in plastic boxes (0.70×0.50×0.18 m, length×width×depth) and their drought tolerance was evaluated. Each box contained 90 seedlings for each type of F3 plants. Three independent replications were performed in a greenhouse using 16-h-light/8-h-dark, 28/22° C. and a room humidity of 60% to obtain the statistical data. Drought was applied to the 10-day-old plants by withholding water. When SWC decreased from 40% to near 0%, and wilting and death of the seedlings were visible, plants were re-watered in order to identify surviving plants. The survival rate of each genotype was recorded. Three replications were carried out for statistical analyses.
The Acquisition of the Protein ZmNAC111 and its Encoding Gene
Corn inbred line B37 seeds were germinated under 28° C. for three days, and then the budding seeds were transferred to soil or solution with nutrients for 3-week cultivation. The whole plant was quick frozen and grinded, and then the total RNA was extracted and subjected to an inverse transcription so as to obtain cDNA. Then PCT amplification was performed with the cDNA as the template and 5′-ATGCCGAGAAGCGGCGGCG-3′ (SEQ ID NO. 29) and 5′-CTACTGCATCCCGATGTGGC-3′ (SEQ ID NO. 30) as the primers. The amplified products were subjected to an agarose gel electrophoresis and 1.4 kb PCR amplification products were obtained. After sequencing, the PCR products have the nucleic acid shown by locus from 157-1584 in SEQ ID NO. 28, the gene is denoted as ZmNAC111; the protein encoded by said gene is called ZmNAC111; and the amino acid sequence of the protein is SEQ ID NO. 27. In SEQ ID NO. 28, locus from 1 to 156 is the 5′ non-coding region, locus 157-1584 is the coding sequence and locus 1585-1824 is the 3′ non-coding region.
Transcriptional Activation Activity Analysis for Different Monomeric Coded Protein ZmNAC111
Drought tolerant corn inbred lines CIMBL55, CIMBL91, CIMBL19, CIMBL22 and CIMBL23 and sensitive corn inbred lines Mo17, D863F, BY4944, SHEN5003, which carry different genotypes, as well as CDNA of corn inbred line B73 serve as the templates.5′-ATGCCGAGAAGCGGCGGCG-3′ (SEQ ID NO. 29) and 5′-CTACTGCATCCCGATGTGGC-3′ (SEQ ID NO. 30) were used as primers for PCR amplification; the target gene clones were incorporated into the yeast expression carrier pGBKT7, and respectively transformed into the yeast strain AH109 (containing the reporter genes HIS3 and ADE2). The transformation of empty vector pGBKT7 served as the reference and recombinant yeast strains ZmNAC111-CIMBL55, ZmNAC111-CIMBL91, ZmNAC111-CIMBL19, ZmNAC111-CIMBL22, ZmNAC111-CIMBL123, ZmNAC111-Mo17, ZmNAC111-D863F, ZmNAC111-BY4944, ZmNAC111-SHEN5003, ZmNAC111-B73 and pGBKT7-Control were obtained respectively. AH109 recombinant yeast strain was coated on the plate of nutrition-deficient medium. Then, the transcription activation activity of ZmNAC111 encoded by different monotypes was compared with the growth of the bacterial plaque.
As shown in
McrBC-Based DNA Methylation Assay.
Genomic DNA was isolated from fresh young leaves collected from B73 and CIMBL55 maize lines before (RLWC=98%) or after drought treatment (RLWC=70%). DNA (1 μg) was digested for 16 hrs at 37° C. with 10 units of McrBC enzyme, a DNA methylation sensitive enzyme (Takara), in parallel with a mock reaction. 50 ng of digested DNA was used for qPCR reactions. DNA hypermethylation was demonstrated by the lower amount of amplification products in the qPCR analysis. All results were obtained by digesting at least two biological replicates and two independent McrBC digests. qPCR was performed using the following conditions: step 1: 95° C., 10 min; step 2: 95° C., 15 sec, 60° C., 30 sec (40 cycles). McrBC digestion at the ZmNac111 gene was normalized to the reference gene maize Ubi-2 and Actin1 and then to the undigested control. Arabidopsis plants were grown on MS agar plates for twenty-one days prior to collection. Actin8 was used as the reference gene in Arabidopsis64. Digestion levels have been inverted to represent methylation levels.
Bisulfite Analysis.
Bisulfite treatment was performed on 200 ng of genomic DNA by using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, Calif.). After bisulfite conversion, the treated DNA was amplified by PCR. Amplified fragments were cloned into the pGEM-T vector (Promega) for sequencing. At least eight clones of each genotype were sequenced.
ChIP Assay.
Fresh young leaves were collected from B73 and CIMBL55 maize lines grown under normal and drought conditions as described above. Whole plants of Arabidopsis were collected from 35S:ZmNAC111-B73 and 35S:ZmNAC111-CIMBL55 lines grown on the MS agar plates for twenty-one days. ChIP assays were performed as previously described65. 7 μl antibodies of anti-H3 (Abcam; ab1791) or anti-H3K9me2 (Abcam; ab1220) were used for the ChIP assays. The amount of immunoprecipitated ZmNac111 chromatin was determined by qPCR on different regions of the ZmNac111 locus. Maize Ubi-2 and Actin8 were used as internal controls for maize and Arabidopsis, respectively. The relative abundance was normalized to the amount of DNA immunoprecipitated by a Histone 3 specific antibody.
Generation Transgenics in the RdDM Mutant Backgrounds.
Two 35S:gZmNAC111-B73-2, -23 and 35S:gZmNAC111-CIMBL55-5, -12 homozygous T2 lines were crossed with five mutants: dcl2-1;dc13-1;dcl4-2 (CS16391), drm1-2;drm2-2 (CS16383), ago4-5 (CS9927), rdr2-2 (SALK_059661), and suvh4-3 (Salk_105816) (http://www.arabidopsis.org). Through PCR-based genotyping analysis, at least three independent F2 homozygous plants for each cross were obtained and harvest. Since the DCL3 locus was heterozygous, only 35S:gZmNAC111-B73-2 and -23 in the dcl2-1;dcl4-2 mutant background were obtained. The ChIP assay was performed using F3 RdDM-mutant homozygous plants obtained from crossing the two independent transgenic Arabidopsis with the RdDM mutants. The F3 plants germinated on Kanamycin-selective medium were used for further DNA methylation and ChIP analyses. Arabidopsis T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Center.
Drought Tolerance of the Transgenic Arabidopsis
The ZmNac111 genomic region in the B73 and CIMBL55 inbred lines, and the coding region in B73, were amplified and inserted into the pGreen vector66 under the CaMV 35S promoter using the NotI and XhoI restriction sites. The constructed plasmid was transformed into the GV3101 Agrobacterium tumefaciens strain containing the pSoup helper plasmid. Arabidopsis thaliana ecotype Col-0 was transformed by Agrobacterium-mediated transformation and independent T2 transgenic lines were obtained using kanamycin-based selection. ZmNac111 gene expression in transgenics was determined by qRT-PCR, in which Actin8 was used as an internal control for normalization. For the drought tolerance assays, seven-day-old plants were transferred into pots containing 250 g of soil. Thirty two-day-old plants growing under favorable water conditions were exposed to drought stress. Water was withheld from the plants for 14 days. Watering was then resumed to allow the plants to recover. Six days later, the number of surviving plants was recorded. At least 64 plants of each line were compared with empty-vector transformed (VC) plants in each test, and statistical data were based on data obtained from three independent experiments.
The Acquisition of ZmNAC111 Arabidopsis
Recombinant tumefaciens X was used to transform the wild type Columbia ecotype Arabidopsis by using flower bud immersion method to obtain T1 generation seeds; T1 generation seeds were screened by using MS medium containing 30 mg/L kanamycin and then the seedlings showing the resistance to kanamycin were cultivated and harvested to obtain T2 generation seeds; T2 generation seeds were screened by using MS medium containing 30 mg/L kanamycin, kanamycin-resistant seedlings showing the kanamycin-resistance segregation ratio of 3:1 were selected to be T2 generation transformed ZmNAC111 Arabidopsis.
The RNA of T2 generation transformed ZmNAC111 Arabidopsis was extracted and was reversely transcribed to obtain cDNA as the template. The cDNA of gene ZmNAC111 was then subjected to PCR amplification with the use of specific primers F1 and R1, wherein the gene of Actin2 of Arabidopsis was an internal reference and the primers were FC and RC.
The sequences of the above-mentioned primers are as follow:
1428 bp positive T2 generation transformed ZmNAC111 Arabidopsis was thus obtained.
Positive T2 generation transformed ZmNAC111 Arabidopsis was harvested to obtain T3 transformed ZmNAC111 Arabidopsis seeds which were then screened by using MS medium containing 30 mg/L kanamycin to obtain 3 homozygosis T3 transformed ZmNAC111 Arabidopsis strains, which were respectively named as TL1, TL2 and TL3 and these strains presented no kanamycin resistance segregation.
A similar method was applied to use the recombinant tumefaciens CK to transform the wild type Columbia ecotype Arabidopsis by flower bud immersion method. Based on the above screening method, homozygosis T3 transformed empty vector Arabidopsis that presents no kanamycin resistance segregation was obtained and named as CK or VC (empty vector).
T1 generation represents seeds obtained from transforming the general generation and plants grown from them; T2 generation represents seeds obtained from transforming T1 and plants grown from them; T3 generation represents seeds obtained from transforming T2 and plants grown from them.
Specific procedures of the above-mentioned flower bud immersion were as follow: Recombinant tumefaciens X or CK was inoculated in LB liquid medium containing 50 mg/L kanamycin and 5 mg/L tetracycline, and was incubated at 28° C. under shaking until OD600 reached 0.8. The mixture was then centrifuged at 25° C., 5000 rpm for 2 minutes to remove the supernatant. The thallus was re-suspended with a re-suspending solution (the solvent was water and the concentration of the solute of sucrose and silwet77 was 50 g/L and 0.02% (volume percentage) respectively) to obtain a dipping solution. The flower bud and the growing points of the plant were dotted with the dipping solution by a pipette and were covered with thin films. After moisturizing the flower bud and the growing points of the plant for 2 days, they then grew under normal conditions for 2 days to harvest seeds.
The above product—T3 transformed ZmNAC111 Arabidopsis strains (TL1-TL3) were extracted. The total RNA of the T3 transformed empty vector Arabidopsis (CK or VC) was obtained and reversely transcribed to obtain cDNA, cDNA served as a template. PCR amplification was performed on cDNA of genes ZmNAC111 with specific primers F1 and R1, wherein the gene of Actin2 of Arabidopsis was an internal reference and the primers were FC and RC.
Electrophoresis results of PCR amplification products are shown in
Drought Tolerant Phenotypic Analysis of Transformed ZmNAC111 Arabidopsis
Plants of 7-day seedling age of the following were obtained: T3 transformed ZmNAC111 Arabidopsis strains (TL1-TL3 or OE6, OE7, OE8), the wild type Arabidopsis (CK or VC) and T3 transformed empty vector Arabidopsis (CK or VC). These plants were transferred to bowls containing 100 g nutrient soil and were allowed to grow under normal conditions for 25 days. After that, these plants were subjected to a drought treatment (i.e. stopping watering). 14 days later, there were obvious differences on the phenotype of the plants, i.e. CK/VC strain rosette leaves were severely dried up while the rosette leaves of TL1-TL3 strains were heavily wilting, these plants were then re-watered. After re-watering for 6 days, statistics for survival rate of each plant in each strain were obtained (plants which grew normally and could be harvested were defined as survival ones; plants which failed to grow normally and could not be harvested, as well as severely influenced by the drought were defined as dead ones; the survival rate is the percentage of the number of survival plants divided by the total number of plants in each strain). The experiment was repeated for 3 times. In each repeated experiment, there were no less than 30 plants in each strain, and the average value was evaluated for the statistics analysis.
Results are shown in Table 2 and
The results of T3 transformed empty vector Arabidopsis (CK or VC) have no remarkable difference with that of the wild type Arabidopsis.
Arabidopsis after drought treatment.
ABA-Sensitivity in the Transgenic Arabidopsis.
The VC and 35S:ZmNAC111 transgenic plants were grown in parallel and harvested. Seeds obtained from these plants were planted on ½×MS plates containing 1% sucrose and were supplemented with or without different concentrations of ABA (0, 0.5. and 1 μM ABA). Plates were chilled at 4° C. in the dark for 5 days for stratification and moved to 22° C. with a 16-h-light/8-h-dark cycle. Germination (emergence of radicals) was scored on the 3rd day after germination, with three replicated assays.
Stomatal aperture assays were conducted as previously described [67]. Briefly, rosette leaf peels were floated in a stomatal opening solution (10 mM MES-Tris, pH 6.15, 100 μM CaCl2), and 10 mM KCl) for 2 hrs and then transferred to a solution supplemented with various concentrations of ABA (0, 0.1, 1, and 10 μM) for another 2 hrs. Subsequently, the abaxial surface of each leaf was applied to 3M clear tape to peel off the epidermal layer. Stomatal apertures were imaged and measured using Image J software. Forty-five stomatal apertures were analyzed in each experiment and the reported values represent the mean±s.d.
The results are shown in
Generation and Analysis of the Transgenic Maize.
In summary, the coding region of ZmNac111 was amplified from B73 and the sequence-confirmed PCR fragment was inserted into the pSBII vector under the control of the Zmubi1 promoter. The pSBI plasmid was then inserted into the LBA4404 A. tumefaciens strain. The LBA4404 strain, with the integrated pSBIII plasmid, was then used to deliver the Zmubi1:ZmNAC111 expression cassette into the A188 maize inbred line as described68. Transgenic T0, T1, and T2 plants were grown in a greenhouse under a 16 h-light/8 h-dark condition. Transgenic positive and the sibling transgenic-negative (WT) plants were determined in each generation by PCR analysis for the transgene. The expression of ZmNac111 in transgenic plants was determined by qRT-PCR. Three independent T2 lines, ZmNAC111-OE1, ZmNAC111-OE3, and ZmNAC111-OE7, were selected for further analyses.
In more detail, the recombinant tumefaciens Y transformed corn inbred line A188 by gene transformation method mediated by tumefaciens to obtain T0 generation plants which grew in a greenhouse (16 hours-illumination/6 hours-darkness).
RNA of T2 transformed ZmNAC111 corn plant was extracted and reversely transcribed to obtain cDNA. PCR identification was performed on cDNA of gene ZmNAC111 with the use of specific primers F2 and R2.
The sequences of the above-mentioned primers are as follow:
1125 bp positive T0 generation transformed ZmNAC111 corn was obtained.
After self-fertilization of the positive T0 generation transformed ZmNAC111 corn, T1 generation seeds were obtained. After using the same method of applying PCR identification on T1 plants, positive plants were obtained. After self-fertilization, T2 seeds were obtained.
The RNA of T2 transformed ZmNAC111 corn was extracted and reversely transcribed to obtain cDNA. qPCR quantification was performed on cDNA of gene ZmNAC111 with the specific primers F2 and R2, wherein the corn gene Zmubi2 was used as an internal reference and the wild type corn was used as the control.
The sequences of the above-mentioned primers are as follow:
The results are as shown in
The above T0 generation represents plants obtained by transforming the present generation; T1 generation represents seeds produced by the self-fertilization of T0 generation and plants grown from the seeds; T2 generation represents seeds produced by the self-fertilization of T1 generation and plants grown from the seeds.
The specific procedures of genetic transformation method mediated by the above-mentioned tumefaciens are as follows:
The recombinant tumefaciens Y was inoculated in YEB liquid medium containing 25 mg/L spectinomycin and incubated at 28° C. with shaking until its OD600 became 0.5. The corn young embryo was placed in 2 mL centrifuge tube filled with a preserving fluid and subjected to a thermal treatment at 46° C. for 3 minutes, followed by centrifugation at 4° C. and 2000 rpm for 10 minutes. The prepared recombinant tumefaciens was then added to the young embryo after treatment, incubated in darkness at 22° C. for 3 days, and then, transferred to a new medium for incubation at darkness, 28° C. for 7 to 10 days. By screening with phosphinothricin at different concentrations, it was finally transferred to a differential medium and subsequently transferred to a rooting medium for cultivation. Once the plant grew into a certain size, it would be transferred to the soil with nutrients.
The recombinant tumefaciens CK1 transformed corn inbred line A188 with the genetic transformation method mediated by tumefaciens, and was cultivated until T2 generation transformed empty vector corn was obtained.
Transgenic-positive (individually genotyped by transgene-based PCR analysis) and WT plants were planted side-by-side in enriched soil (soil and vermiculite in a ratio of 1:1). Drought treatment was applied to the soil-grown plants at the 3-leaf seedling stage by withholding water. After approximately 20 days, watering was resumed to allow plants to recover. The number of surviving plants was recorded seven days later. At least 15 plants of each line were compared in each test and statistical analyses were based on data obtained from three independent experiments. Stomatal conductance, photosynthetic and transpiration rates of the T2 transgenic and WT plants were measured on fully-expended leaves of seedlings at the 3-leaf stage using a LICOR-6400 CO2 gas exchange analyzer (LICOR-6400, Lincoln, Nebr.). SWC was recorded every other day after the initiation of water withholding. Statistical analysis was based on data obtained from seven seedlings for each plant line and the experiment was repeated twice.
Results are shown in Table 3 and
Photosynthesis Analysis for the Transformed ZmNAC111 Corn
Net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were measured with Li6400 portable photosynthesis system (LICOR-6400, Lincoln, Nebr.). In the third-leaf stage, the third unfolded leaf of T2 generation transformed ZmNAC111 corn strains (TML1-TML3 (OE1, OE3, OE7)) and the wild type corn plants (WT) were measured and subjected to a treatment of no watering, which were then measured once every two days. The corresponding water content in the soil was recorded. 7 plants were randomly measured in each transformation time and the measurement was repeated for 2 times. The measured results are expressed by mean values.
The results are shown in
RNA-Seq Analysis of Transgenic Maize.
For maize RNA-seq analysis, pooled tissues from three eight-day-old maize seedlings were collected from transgenic and WT plants, prior to or after 2-hour dehydration on a clean bench, to conduct the RNA-seq analysis. Total RNA was isolated using TRIZOL reagent (Biotopped) and RNA integrity was evaluated using a Bioanalyzer 2100 (Agilent). The 100-bp paired-end Illumina sequencing was conducted at Berry Genomics (Beijing). An average of 3 gigabases of raw data were generated for each sample. Differential gene expression was determined using Strand NGS 2.0 software. A total of 31,501 genes were identified, representing ˜79% of all the predicted genes in maize. Enrichment analysis of gene ontology of biological pathways (GOBPs) was performed using the DAVID software program69 (http://david.abcc.ncifcrf.gov/) to compute P-values that indicate the significance of each GOBP being represented by the genes. GOBPs with P<0.01 were identified as enriched processes. qRT-PCR of selected genes that were determined to be critical to drought tolerance was performed to verify the RNA-seq data.
RNA-Seq Analysis of Transformed ZmNAC111 Arabidopsis
20-day aged plants of the following were obtained: the transformed ZmNAC111 Arabidopsis strains (TL1-TL3) and T3 transformed empty vector Arabidopsis strain (CK). They were respectively subjected to drying for 0 hr and 1 hr, wherein each strain had at least 10 seedlings. The total RNA was isolated by TRIZOL (Biotopped) method and the concentration was measured by using Nanodrop1000 (Thermo Scientific product, USA). Then, the total RNA was sent to BerryGenomic Corporation in Beijing to perform transcriptome profiling with a sequencing depth of 3 GB. Lastly, the data was evaluated with Strand NGS 2.0 software
Nucleotide Diversity and Tests for Neutrality.
The genomic region of ZmNac111 was amplified and sequenced in 42 teosinte accessions. Nucleotide diversity (Tr) and the Tajima's D-statistic were calculated using DnaSP version 5.070.
Phylogenetic Tree Construction.
The full-length amino acid sequences of 55 NAC TF encoding genes identified in maize, rice, Arabidopsis, and sorghum were aligned using the Clustal X 1.83 program with default parameters. The phylogenetic tree was constructed based on this alignment result using the neighbor joining (NJ) method in MEGA version 5 with the following parameters: Poisson correction, pairwise deletion, uniform rates and bootstrap (1000 replicates).
Transactivation Activity Assay.
cDNAs of ZmNac111 from ten maize inbred lines were individually cloned into the pGBKT7 vector for evaluating protein transactivation activity in the AH109 yeast strain. The cell concentration of yeast transformants was adjusted to an OD600 of 0.1, and then plated on various selective plates, SD/-T, SD/-T-H, SD/-T-H-A, to compare their survival. Plates were incubated at 30° C. for 2-5 days before photographing. All the primers used in this research are listed in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201510437994.8 | Jul 2015 | CN | national |
| PCT/CN2015/090084 | Sep 2015 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2016/052220 | 7/22/2016 | WO | 00 |
