Patent Application
20110119790
The present invention relates to a gene participating in a low temperature germinability and a technique for utilizing the same. More specifically, this invention relates to an isolated gene for low temperature germinability having the ability to improve the low temperature germinability in rice, the amino acid sequence encoded by the gene, a method of improving germinability under environmental stress (the stresses of low temperature, salt, and osmotic pressure) using the gene for the low temperature germinability, a transgenic plant transformed with the gene, and a method of detecting low temperature germinability in plants.
Resistance to environmental stress is an important trait in crops, and has until now been thought to be a complex trait controlled by a plurality of genes called quantitative trait loci (QTLs). Identifying these QTLs is believed to be important for achieving stable crop production in the world. The inventors have previously disclosed a method of screening gramineous plants having specific traits, and gene markers for use in such a method (Patent Document 1). In the course of conducting research on the low temperature germinability in rice, the inventors have also found, through QTL analysis of genes for germinability in rice, three QTLs, qLTG-3-1, qLTG-3-2, qLTG-4, on chromosome 3 and chromosome 4. The locus qTLG-3-1 which was found at the end of the short arm of chromosome 3 has a very large activity, from which it was concluded that it might be useful for improving the low temperature germinability in rice (Non-Patent Document 1).
In addition, of the three QTLs which act on the low temperature resistance at the germination stage, the inventors have carried out fine mapping on qLTG-3-1 (Non-Patent Documents 2 and 4). As a result, they have succeeded in narrowing the candidate regions for qLTG-3-1 to the approximately 96-kb region between the marker SSR125411-4.1 and the marker STS73-28 (Non-Patent Document 2). It was possible, from the fine mapping of QTLs controlling low temperature germinability, to infer this to be a candidate region for qLTG-3-1.
The inventors have also carried out QTL analyses on the seed germinability in rice under stress. The results suggest that qLTG-3-1 participates in responses to diverse stresses, including temperature, salt (NaCl), and osmotic pressure (mannitol) stress. In this QTL analysis, it became clear that a Italica Livorno gene exhibits a germinability-increasing activity and that a QTL which exhibits a high germinability to both salt and osmotic pressure stresses exists at the end of the short arm of chromosome 3 (Non-Patent Document 3).
Thus, low temperatures are a major environmental stress in world crop production, and the inventors have hitherto found, by means of QTL analysis using Italica Livorno, which has a high low-temperature germinability, three QTLs (quantitative trait loci) which participate in low temperature germinability. However, environmental stresses had been thought to be complex traits controlled by a plurality of genes, the genetic function of the qLTG-3-1 phenotype had remained to be clarified, and the qLTG-3-1 gene had yet to be isolated, identified or cloned.
In light of these circumstances, the inventors have reflected on the above prior art and conducted extensive investigations with the aim of elucidating the genetic function of the qLTG-3-1 phenotype, isolating and identifying the qLTG-3-1 gene, and creating transformants by cloning this gene. In the course of these investigations, to elucidate the above QTL at the molecular level, the inventors have identified qLTG-3-1 by means of chromosome map-based cloning and discovered that this gene encodes proteins of unknown function. Also, qLTG-3-1 is strongly expressed in the embryo at the time of seed germination and, in transgenic plants obtained by means of a qLTG-3-1 promoter fused to GUS, distinctive GUS staining was observed in the bud scales and ventral scales which cover the coleoptile and seminal roots. The inventors also conducted further studies, in the course of which they succeeded in elucidating the function of qLTG-3-1, determining the base sequence of the qLTG-3-1 gene, determining the amino acid sequence encoded by this gene, creating transformants with the gene, and developing techniques for utilizing the gene.
Accordingly, the objects of the present invention are to provide a gene participating in low temperature germinability in rice, an amino acid sequence encoded by the gene, a method of improving germinability under environmental stresses (the stresses of low temperature, salt, osmotic pressure) that makes use of the gene, transformants created by the introduction of the gene, and a method of detecting the low temperature germinability in plants.
The present invention for solving the above problems is constituted of the following technical means.
Next, the present invention is described in greater detail.
The present invention provides a gene for a low temperature germinability which is an isolated qLTG-3-1 gene from the rice line Italica Livorno, has low temperature germinability, and is characterized by having the base sequence of SEQ ID NO: 1 in the sequence listing. The preferred embodiments of the present invention are that the above gene for low temperature germinability has a genetic mutation present in a portion of the base sequence of SEQ ID NO: 1 and has a low temperature germinability identical or similar to that of the base sequence of SEQ ID NO: 1, the genetic mutation is due to the addition, deletion or substitution of a base sequence, and the above gene for low temperature germinability has a germinability-improving function under environmental stresses of low temperature, salt (NaCl) and osmotic pressure (mannitol).
The present invention also provides an amino acid sequence which is encoded by the above gene for low temperature germinability, has a low temperature germinability-improving action, and has the amino acid sequence of SEQ ID NO: 20 in the sequence listing. The present invention also provides a transgenic plant obtained by recombination of the above gene for low temperature germinability in a plant to improve the low temperature germinability thereof. In one preferred embodiment of the transgenic plant of the present invention, the plant is rice. In another preferred embodiment, the qLTG-3-1 promoter +qLTG-3-1 gene has been introduced into the plant to improve the low temperature germinability. In yet another preferred embodiment, the 35S promoter +qLTG-3-1 gene has been introduced into the plant to induce overexpression of gene qLTG-3-1 for the low temperature germinability.
The present invention also provides a method of analyzing low temperature germinability, comprising analyzing the low temperature germinability in a cultivar by comparing the base sequence of the above gene for low temperature germinability with the genotype of the cultivar. In a preferred embodiment of the method of analysis of the present invention, the cultivar is rice. In addition, the present invention also provides a method of improving the low temperature germinability in rice, comprising introducing the above gene for low temperature germinability into a rice cultivar so as to improve the low temperature germinability in the cultivar under low temperature conditions.
Next, the isolation of the gene for the low temperature germinability of the present invention is described in detail. The plant materials used were Hayamasari, a rice originated in Japan that is a Japonica rice variety, and Italica Livorno, a rice originated in Italy. With regard to NILHYqLTG-3-1, which is a near-isogenic line (NIL) for qLTG-3-1, a 360-kb chromosomal region near qLTG-3-1 from Italica Livorno was introduced into Hayamasari. Based on the phenotype and genotype of low temperature germinability, BIL116 was screened from the recombinant self-fertile line BILs obtained by crossing Hayamasari with Italica Livorno. BIL116 was backcrossed with Hayamasari by marker-assisted screening in order to create NIL.
To study the germinability under difference stress conditions, germination tests were carried out according to the method reported by Fujino et al. (2004). For low-temperature stress, seeds were placed on Petri dishes which were then loaded into an incubator. Solutions of plant hormones (ABA and GA), NaCl and mannitol at different concentrations were added to the Petri dishes which were then placed in an incubator.
The fine mapping and high-resolution mapping of qLTG-3-1 were performed in order to carry out the positional cloning of qLTG-3-1. Molecular markers and polymorphism between both parents mentioned in subsequent Table 1 were used for this purpose. BIL116 was crossed with Hayamasari, and backcross progeny were created for the sake of fine mapping and high-resolution mapping. An F2 population composed of 256 individuals was used for fine mapping.
The genotype of each recombinant F2 individual at the qLTG-3-1 locus was determined by germination testing the F3 progeny at a low temperature. The three genotypes—homozygous for the Italica Livorno allele, homozygous for the Hayamasari allele, and heterozygous—were clearly distinguishable. These F2 individuals were used in fine mapping of qLTG-3-1, in addition to which a BC1F2 population composed of about 3,200 individuals was used in high-resolution mapping.
The genomic DNA was extracted according to the method reported by Fujino et al. (2004). The genomic DNA was used in a polymerase chain reaction (PCR) for the purpose of cosegregation with a molecular marker-bearing phenotype based on PCR. In addition to the two SSR markers developed thus far (Fujino et al., 2004), using the Nipponbare genome, eight SSR markers (see subsequent Table 1) were prepared for fine mapping according to the method reported by Fujino et al. (2004).
For high-resolution mapping, six molecular markers were created based on 14 differences in the genomic sequence between Hayamasari and Italica Livorno (see subsequent Tables 1 and 2). For sequencing to detect polymorphisms between the parents in the 96-kb region, both strands of the PCR product from the parents were directly sequenced using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).
For association analysis, 69 Japonica rice varieties were classified based on the qLTG-3-1 genotype. To detect the Hayamasari allele, PCR which amplifies deleted regions found in Hayamasari was carried out using the primers S103U and S103L (see subsequent Table 4). To detect the Nipponbare allele, the above-mentioned PCR product was digested with BseRI. The base sequence GAGGAG in the Italica Livorno allele was digested with BseRI, and the base sequence GTGGAG in the Nipponbare allele was digested with BseRI. Genotypes of Hayamasari, Nipponbare and Italica Livorno were found in, respectively, 28, 20 and 21 rice varieties. The mean low temperature germinabilities of the respective genotypes were compared by analysis of variance.
To carry out an analysis of gene expression, the total RNA was extracted from various organs of rice using RNAiso (TAKARA). The total RNA (0.5 μg) was reverse-transcripted with Rever Tra Ace (TOYOBO) having an Oligo(dT)20 primer, in accordance with the manufacturing guidelines. The PCR reaction was carried out using KOD-plus (TOYOBO). Each PCR reaction (10 μL) included 0.5 μL of cDNA template that had been diluted 5-fold. The specificity of each primer for the target gene was confirmed by PCR product sequencing. For the purpose of Northern blotting analysis, the total RNA (4 μg/sample) was isolated on 2.0% (w/v) agarose-modified formaldehyde gel containing 40 mM MOPS (pH 7.0), 10 mM Na-acetate, 1 mM EDTA and2% (v/v) formaldehyde.
The RNA was transferred with 20×SSC to a positively charged nylon membrane (Roche Diagnostics). Hybridization and signal detection were carried out by, respectively, a DIG system and CDP-star (Roche Diagnostics). The PCR fragments from the primers 13-5U and 13-5L were used as the probes for Northern blot analysis. The primers and amplification conditions for RT-PCR analysis are shown subsequently in Table 5.
To carry out plasmid construction and transformation, a 3-kb genomic DNA fragment of qLTG-3-1 from Italica Livorno was amplified by PCR using the primers Ano13-LA5U and Ano13-LA5L (see subsequent Table 4). To create a qLTG-3-1 promoter GUS gene fusion construct, a 2-kb genomic DNA fragment of the 5′ upstream region of qLTG-3-1 from Italica Livorno was amplified by PCR using the primers Ano13-10U and Ano13-10L (see subsequent Table 4).
Because a promoter sequence that fully expresses the true qLTG-3-1 gene has yet to be identified, a 2k-bp 5′ upstream region from the initiation codon of qLTG-3-1 was used as the promoter. These PCR products were cloned to, respectively, the BamHI/SacI and HindIII/BamHI sites of the pBluescript II SK vector (Stratagene).
Next, these fragments and the GUS gene were cloned to the pPZP2H-lac Ti-plasmid vector (Fuse et al., 2001). To produce an overexpression plant, a construct in which the qLTG-3-1 gene was ligated below the 35S promoter was created. PCR products from the primers Ano13-LA5U and Ano13-LA5L were digested with BamHI and SacI. These fragments were cloned at the Sad site of the pPZP2Ha3 Ti-plasmid vector (Fuse et al., 2001).
An Agrobacterium-mediated transformant was used to transform Hayamasari (Toki, 1997; Toki et al., 2006). A plant regenerated from a hygromycin-resistant callus (T0 plant) was grown in an isolated greenhouse. The T1 transformant was subjected to selection by PCR on the transgene, cultivation and collection (T2 generation), and furnished to germination experiments.
To carry out the histochemical analysis of GUS expression, the seeds of transgenic plants having qLTG-3-1::GUS were incubated at 30° C. Under these conditions, germination (emergence of the coleoptile) began to arise in a very small percentage of the seeds two days after treatment. The seeds of the transformants were collected as specimens 0, 1 and 2 days after treatment. All the seeds of transgenic rice and longitudinally cut seeds were vacuum immersed in 50 mM NaH2PO4 (pH 7.0) containing 0.5 mM X-Gluc, 0.5 mM K3(Fe(CN)6), 0.5 mM K4(Fe(CN)6) and 0.5% (v/v) Triton X-100, and incubated at 37° C. for 6 hours. Next, 70% EtOH was added thereto to halt the enzyme reaction at room temperature.
Rice seed of the AA genome wild rice species W0106 (O. rufipogon), W0652 (O. barthii), W1169 (O. glumaepatula), W1413 (O. longistaminata) and W1508 (O. longistaminata) were acquired from the National Institute of Genetics of the Research Organization of Information and Systems as wild relatives of cultivated rice. The seeds of a core collection of cultivated rice (O. sativa) containing 62 varieties (Kojima et al., 2005) were acquired from the National Institute of Agrobiological Sciences.
This collection was composed of three groups, Groups A, B and C, which correspond to Japonica, Aus and Indica (Kojima et al., 2005). The total DNA was isolated from young leaves thereof, using CTAB, by the method reported by Fujino et al. (2004). The qLTG-3-1 gene region was amplified using primers (see subsequent Table 4), then directly sequenced using cycle sequencing with a Big Dye Terminator (Applied Biosystems).
Sequencing was carried out with a Prism 3700 automated sequencer (Applied Biosystems). Alignment of the DNA sequences was carried out using BioEdit (http:www.mbio.ncsu.edu/BioEdit/bioedit.html), following which the sequences were visually confirmed. All polymorphisms were rechecked from chromatograms.
In the sequences found by the inventors, heterozygotes were not observed. A 1,784-bp gene in the qLTG-3-1 gene region that includes a 933-bp upstream region containing 5′ and 3′UTR and a 296-bp downstream region, excluding TA repeats in the 50-bp region from base pairs 433 to 384, was sequenced from the cultivated rices and the wild varieties.
A haplotype network showing unique DNA sequences obtained from gene polymorphism or lineage relationships between alleles was constructed by the computer program TCS (Crandall et al., 2000) using the parsimony method in statistics.
Conferring resistance to environment stresses is an important breeding objective in the stable production of crops. In rice cultivation within cold-weather regions, low temperatures are a major stress. Up until now, low-temperature resistance had been thought to be a complex trait controlled by a plurality of genes called quantitative trait loci (QTLs). This has been addressed in the present invention by isolating genes which are quantitative trait loci and have a large gene activity associated with the low temperature germinability in rice, and carrying out functional analysis on these genes.
The quantitative trait locus qLTG-3-1 in the rice line Italica Livorno having an excellent low temperature germinability has been isolated as a gene for a low temperature germinability. In the present invention, this qLTG-3-1 was molecularly identified by the positional cloning method. This gene was composed of 555 base pairs, and was a novel gene of unknown function. When a functional gene was introduced into the rice strain Hayamasari having a nonfunctional gene by an Agrobacterium method, a low temperature germinability higher than that of Hayamasari was exhibited, from which it became apparent that this 555-bp sequence was the target gene qLTG-3-1 for low temperature germinability.
A near-isogenic line obtained by using DNA marker screening and backcrossing to introduce the chromosomal region containing the functional gene qLTG-3-1 of Italica Livorno into the rice variety Hayamasari having a nonfunctional gene exhibited a high germinability not only at low temperatures, but also under salt (NaCl) and osmotic pressure (mannitol) stresses. This suggests that qLTG-3-1 is a gene for resistance to a plurality of stresses.
As a result of gene expression analysis, high expression of qLTG-3-1 was confirmed, particularly in embryos at the time of seed germination. Moreover, high expression was also observed in the panicles prior to panicle emergence. The GUS gene was thus ligated to a 2-kb upstream region on the 5′ side of the initiation codon, and this construct was introduced by the Agrobacterium method into Hayamasari. As a result, GUS activity was observed in the seed embryos at the time of germination. It became clear from these observations that qLTG-3-1 is specifically expressed in seed embryos during germination, and that this specificity is controlled by at least a 2-kb upstream region on the 5′ side.
On comparing the gene sequences of the gene qLTG-3-1 for the low temperature germinability, it was found that Italica Livorno, which exhibits a high low-temperature germinability, had a functional gene sequence, whereas Hayamasari had lost functionality due to the deletion of some 71 base pairs. Nipponbare had a single base substitution that gives rise to an amino acid mutation. Hence, an association analysis on the qLTG-3-1 genotype and low temperature germinability was carried out for about 70 rice varieties.
As a result, the following relationship in low temperature germinability was clearly observed among the three lines: Italica Livorno>Nipponbare>Hayamasari. It was apparent from this that the low temperature germinability can be inferred by discerning gene mutations. Also, the mutated amino acid observed in Nipponbare was thought to be related to the function of the gene. The region containing this mutated amino acid was highly conserved even in genes homologous to qLTG-3-1 in plants.
In the present invention, to create a marker for narrowing the candidate region of qLTG-3-1, an 86-kb candidate region in Italica Livorno and a 90-kb candidate region in Hayamasari were PCR amplified based on the Nipponbare sequence, and sequence analysis was carried out. As a result, mutations at 14 places were identified; six of these were rendered into markers and used to screen recombinant individuals from a large-scale population. About 3,200 individuals were furnished for testing; it was possible with these to narrow the candidate region for qLTG-3-1 to the 4.8-kb between markers D and F. Using the Rice Annotation Database (RAP-DB), it was predicted that one gene of unknown function is present in this region.
Compared with the functional gene of Italica Livorno, a 71-bp deletion arose in Hayamasari, which clearly resulted in a loss of function. When the functional gene of Italica Livorno was introduced into Hayamasari, the transformant T2 clearly exhibited a higher low-temperature germinability than Hayamasari. It was thus possible to confirm that this is the gene responsible for gene qLTG-3-1 for the low temperature germinability.
The expression of the qLTG-3-1 gene was observed to have a high tissue specificity. Gene expression was especially strong in embryos during seed germination; expression was not observed in the endosperm. Because expression of this gene is observed even during germination under both 30° C. and 15° C. conditions, this is not a gene that is induced by low-temperature stress. Moreover, given that expression is not observed even in the embryo portion during ripening and that expression rises with elapsed time following germination treatment, it was apparent that qLTG-3-1 exhibits specific gene expression at the time of germination.
Expression of the qLTG-3-1 synthesis genes (OsGA20ox1, OsGA20ox2, OsGA3ox2) and the amylase gene (Ramy 1A) during germination was analyzed using Italica Livorno having the qLTG-3-1 functional gene. Gene expression by qLTG-3-1 was observed 6 hours after treatment at 30° C., and 12 hours after treatment at 15° C. At 30° C., gene expression by OsGA20ox1, OsGA3ox2 and Ramy 1A was similarly observed after 6 hours. On the other hand, at 15° C., gene expression by OsGA20ox1 was observed after 12 hours, as in the case of qLTG-3-1, and gene expression by OsGA3ox2 and Ramy 1A was observed after 24 hours. It was apparent from this that the expression of these genes begins at about the same time as gene expression by qLTG-3-1.
Such a gene expression pattern was also observed in the Hayamasari near-isogenic line NILHYqLTG-3-1 for the qLTG-3-1 functional gene from Italica Livorno. Also, in Hayamasari having the qLTG-3-1 nonfunctional gene, the expression of all these genes was delayed. Based on this, it appears that the expression of these genes is induced at an early stage by qLTG-3-1, resulting in a high low-temperature germinability being expressed as a trait.
The responsiveness of qLTG-3-1 to environmental stresses other than low temperatures was analyzed using NILHYqLTG-3-1. As a result, an increase in germinability was observed also in the presence of NaCl and mannitol. Also, in the presence of ABA, the germination of Italica Livorno and Hayamasari was delayed, whereas in the presence of NILHYqLTG-3-1, germination was inhibited.
In addition, by means of this invention, mutations of the qLTG-3-1 gene were elucidated. Hayamasari contained a nonfunctional gene due to a 71-bp deletion with respect to the functional gene qLTG-3-1 of Italica Livorno. In Nipponbare, a single base substitution accompanied by an amino acid mutation arose with respect to the functional gene. As a result of database analysis using qLTG-3-1 base sequences, homologous genes were present in the following plant families: Poaceae, Solanaceae, Leguminosae and Cucurbitaceae. When these amino acid sequences were compared, a highly conserved sequence was detected at the N-terminus (8 amino acid sequences). The amino acid mutation that arose in Nipponbare took place within this conserved sequence.
Here, in order to clarify the function of the Nipponbare type qLTG-3-1 gene, an association analysis was carried out using 69 rice lines ranging from varieties native to Hokkaido to present-day varieties. As a result, clear differences were observed in the qLTG-3-1 genotypes and low temperature germinabilities in these lines. The germination rate was 80.8% among Italica Livorno rices (21 lines), 69.7% among Nipponbare rices (20 lines), and 30.7% among Hayamasari rices (28 lines); hence, significant differences were obtained. These results showed that the amino acid mutation which arose in Nipponbare lowers somewhat the gene function of qLTG-3-1. In addition, the conserved amino acid sequence was also thought to have an important role in gene function.
To search for the qLTG-3-1 genotype mutations in cultivated rice, the 1784-bp gene sequence was compared for 62 lines from the world core collection. As a result, in-frame insertions and deletions were noted at three places in the structural gene region, but no amino acid substitutions were present. Such insertions and deletions gave rise to differences in the number of repeats within the reiterated sequence region. These results showed that qLTG-3-1 is functionally important; hence, it is thought to be a highly conserved gene. Also, on the basis of these base substitutions, the core collection was made up of two haplogroups of 10 haplotypes.
The following effects are achieved by the present invention.
Next, the present invention is described more concretely by means of examples. However, it is to be understand that these examples do not limit in any way the present invention.
In this example, a genetic assessment of qLTG-3-1 was carried out. The plant materials used were Hayamasari, a rice originated in Japan that is a Japonica rice variety, and Italica Livorno, a rice originated in Italy. Based on the phenotype of low temperature germinability and the genotype of qLTG-3-1, BIL116 was screened from recombinant self-fertile lines (BILs) obtained by crossing Hayamasari and Italica Livorno.
BIL116 was backcrossed with Hayamasari by DNA marker screening in order to create a near-isogenic line (NIL), thereby forming a population of BC3F1 individuals. Of the 66 BC3F1 plants, #59 having the smallest portion of the genome that included qLTG-3-1 from the donor Italica Livorno, was backcrossed. In addition, the genotype of the entire genome of #59 was examined. Based on this, a single individual out of the eleven #59 individuals from the BC3F1 population was selected: #59-11.
Of the BC3F2 progeny obtained by self-fertilization of BC3F1 individual #59-11, those individuals homozygous for the Italica Livorno allele in the qLTG-3-1 region were selected (B in
To date, the inventors have mapped three QTLs that control low temperature germinability by backcrossing recombinant self-fertile lines (BILs) obtained by crossing the low temperature germinating Japonica strain Italica Livorno with ordinary Hayamasari (Fujino et al., 2004). Of these, qLTG-3-1, the QTL thought to be the most effective, was mapped on chromosome 3. To determine the genetic basis of qLTG-3-1, segregation analysis of the low temperature germinability was carried out using the backcross progeny.
The frequency distribution of low temperature germinability in the backcross population had a clear single-factor segregation pattern (
In this example, the isolation of qLTG-3-1 was carried out. Molecular markers and the polymorphisms between both parents shown in Table 1 were used for the fine mapping and the high-resolution mapping of qLTG-3-1. BIL116 was crossed with Hayamasari, and the backcross progeny were bred for the purpose of fine mapping and high-resolution mapping. An F2 population composed of 256 individuals was used for fine mapping. The genotypes of each of the recombinant F2 plants at the qLTG-3-1 gene locus were determined by low temperature germination tests of their F3 progeny. The three genotypes—homozygous for the Italica Livorno allele, homozygous for the Hayamasari allele, and heterozygous—were clearly differentiated.
These recombinant F2 plants were used for the fine mapping of qLTG-3-1. In addition, a BC1F2 population of about 3,200 plants was used for high-resolution mapping. The genomic DNA was extracted according to the method described by Fujino et al. (2004). The genomic DNA was used in PCR analysis; based on the PCR, the phenotype cosegregated with the molecular markers.
In addition to the two SSR markers developed thus far (Fujino et al., 2004), the Nipponbare genome was used to create eight SSR markers (Table 1), according to the method described by Fujino et al. (2004), for the purpose of fine mapping.
For high-resolution mapping, six molecular markers were created (Tables 1 and 2) based on differences in 14 genomic sequences between Hayamasari and Italica Livorno. For sequencing to find polymorphisms between the parents in a 96-kg region, both strands of PCR products obtained from the parents were directly sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).
The 69 Japonica varieties were classified according to the qLTG-3-1 genotype. To find the allelomorphs of Hayamasari, the primers S103aU and S103aL were used to carry out PCR so as to amplify deletions discovered in Hayamasari. The above PCR products were digested with BseRI in order to find the allelomorphs of Nipponbare.
GAGGAG in the Italica Livorno allelomorph was digested by BseRI, but GTGGAG in Nipponbare was not digested. The genotypes of Hayamasari, Nipponbare and Italica Livorno were found in, respectively, 28, 20 and 21 rice varieties. The average low temperature germination rates of the respective genotypes were compared by analysis of variance.
Fine mapping of the genes was carried out using the backcross progeny. qLTG-3-1 was positioned in the 96-kb region between the markers SSR125411-4.1(A) and STS73-28(1).
Six of these were used in high-resolution mapping. As a result, qLTG-3-1 cosegregated with S103a(E), and a 4.8-kb region was found between the markers SSR118673-13.1(D) and S107(F). A single gene, Os03g0103300, was predicted by RAP-DB (http://rapdb.nig.ac.jp/index.html).
On comparison with the qLTG-3-1 sequence in Italica Livorno, a 71-bp deletion was detected in the coding sequence for Hayamasari. This gave rise to a frame shift that created a stop codon. This fact indicates that the allelomorph of qLTG-3-1 in Hayamasari undergoes a loss of gene function. In Nipponbare, it is predicted that mutations of T to A at more than 50 positions in the coding region will convert Leu to His.
By means of a database search, 21 genes having significant homology to qLTG-3-1 were discovered only in plants. By phylogenetic analysis of these proteins which include qLTG-3-1, it was found that these proteins can be divided into two main classes.
In the Nipponbare sequence, the Leu residue at the 9 position on the converted amino acid mutated to His. To determine the function of the converted amino acid, an association analysis was carried out between the genotype of qLTG-3-1 and the phenotype of low temperature germinability. A total of 69 rice varieties were classified into the three qLTG-3-1 genotypes (Table 3,
In this example, complementation testing was carried out. A 3-kb genomic DNA fragment of qLTG-3-1 from Italica Livorno was amplified by PCR using the primers Ano13-LA5U and Ano13-LA5L (Table 4). This PCR product was cloned at the BamHI/SacI site of the pBluescriptIISK-vector (Stratagene). Next, this fragment was cloned at the pPZP2H-lac Ti-Plasmid vector (Fuse et al., 2001).
A plant regenerated from a hygromycin-resistant callus (T0 plant) was grown in an isolated greenhouse. T1 plants were obtained from self-fertile T0 plants. The T1 transformants were selected by PCR on the transgene, and the T2 seeds were collected in order to carry out germination experiments.
For the sake of complementation testing, a 3-kb fragment of Italica Livorno containing the qLTG-3-1 promoter region and the gene region was introduced into Hayamasari by an Agrobacterium-mediated transformant. The transgenic line having the vector exhibited a low temperature germination rate similar to that of Hayamasari. All five transformants that were homozygous for the transgene had higher low-temperature germination rates than Hayamasari (
In this example, the expression of the qLTG-3-1 gene was studied. Using RNAiso (TAKARA), the total RNA was extracted from each organ of rice and treated with DNaseI (TAKARA). For Northern blot analysis, the total RNA (4 μg/sample) was separated on 2.0% (w/v) agarose-modified formaldehyde gel containing 40 mM of MOPS (pH 7.0), 10 mM of sodium acetate and 2% (v/v) formaldehyde.
The RNA was blotted with 20×SSC to a positively charged nylon membrane (Roche Diagnostics). Hybridization and signal detection were carried out with a DIG system and CDP-Star (Roche Diagnostics) according to the manufacturing guidelines. The PCR fragments obtained from the primers 13-5U and 13-5L were used as the probes for Northern blot analysis (Table 5).
qTLG-3-1 was germination treated by Northern blot analysis with Italica Livorno and NILHYqLTG-3-1 at 30° C. and 15° C. in each case, after which expression by the gene occurred at 12 hours and 1 day. Expression increased further with the start of germination (
A physiological assessment of qLTG-3-1 was carried out in this example. Germination tests were carried out by the method reported by Fujino et al. (2004). For the low temperature stress, seeds on a Petri dish were placed in an incubator. Solutions of plant hormone (ABA and GA) and mannitol at different concentrations were added to the Petri dishes at this time before placing the dishes in the incubator.
Endogenous ABA and GA play a major role in promoting seed dormancy and germination (Leung and Girandat, 1998). qLTG-3-1 was studied to determine whether it influences the responses to ABA and GA in the germination period.
At the optimal temperature (25° C.), very slight differences in germination rate were observed between Hayamasari, Italica Livorno and NILHYqLTG-3-1 (
At concentrations lower than 250 mM mannitol and 150 mM NaCl, all the varieties showed a delay in germination (
In this example, the association between qLTG-3-1 expression and seed germination was examined. Total RNA (0.5 μg) was reverse-transcripted by means of ReverTra Ace (TOYOBO) having an Oligo(dT)20 primer, according to the manufacturing guidelines. PCR reactions were carried using KOD-Plus (TOYOBO). Each PCR reaction (10 μL) included 0.5 μL of cDNA template that had been diluted 5-fold. The specificity of each primer for the target gene was confirmed by sequencing the PCR product. The primer and amplification conditions for RT-PCT analysis are shown in Table 5.
The expression of qLTG-3-1 in embryos at the time of Italica Livorno, Hayamasari and NILHYqLTG-3-1 seed germination was determined by RT-PCR analysis. As shown in
The qLTG-3-1 expression pattern was investigated in embryos treated with ABA (500 mM), NaCl (250 mM) and mannitol (500 mM). The delay and suppression of qLTG-3-1 expression was observed (
In this example, a histochemical analysis of qLTG-3-1 expression was carried out. To create a qLTG-3-1 promoter-GUS gene fusion construct, a 2-kb genomic DNA fragment of the 5′ upstream region of qLTG-3-1 from Italica Livorno was amplified by PCR using the primers Ano13-10U and Ano13-10L (Table 4).
Because a promoter sequence that fully expresses the true qLTG-3-1 gene is unknown, a 2-kb 5′ upstream region from the initiation codon of qLTG-3-1 was used as the promoter. This PCR product was cloned to the HindIII/BamHI site of the pBluescript II SK vector (Stratagene). Next, this fragment and the GUS gene were cloned to the pPZP2H-lac Ti-plasmid vector (Fuse et al., 2001). The qLTG-3-1::GUS construct is shown in
An Agrobacterium-mediated transformant was used to transform Hayamasari (Toki, 1997; Toki et al., 2006). Plants regenerated from hygromycin-resistant calluses (T0 plants) were grown in an isolated greenhouse. Self-fertile seeds of each T0 plant (T1) were used in this experiment. The T0 transformants were screened for transgenes by PCR.
To carry out the histochemical analysis of GUS expression, the seeds of transgenic plants containing the qLTG-3-1::GUS construct were incubated at 30° C. Under these conditions, germination and the emergence of the coleoptile began to arise in a very small proportion of the seeds one day after treatment. The seeds of the transformants were furnished for testing 0, 1 and 2 days after treatment.
All the seeds of the transformant and longitudinally cut seeds were vacuum immersed in 50 mM of NaH2PO4 (pH 7.0) containing 0.5 mM X-Gluc, 0.5 mM K3[Fe(CN)6], 0.5 mM K4[Fe(CN)6] and 0.5% (v/v) Triton X-100, and incubated at 37° C. for 6 hours. Next, 70% EtOH was added thereto to stop the enzyme reaction under the temperature.
The qLTG-3-1 promoter activity in transgenic rice plants having the qLTG-3-1::GUS (beta-glucuronidase) reporter-gene fusion construct was analyzed. GUS expression was strongly detected in the bud scales and ventral scales surrounding the seminal roots (
In this example, the induction of GA biosynthesis gene and Ramy 1A expression by qLTG-3-1 was examined. It is not clear whether qLTG-3-1, which is strongly expressed in embryos at the time of seed germination, exhibits a high germination rate under diverse stress conditions. To assess the function of qLTG-3-1, the expression profiles of six GA biosynthesis genes and the α-amylase gene Ramy 1A, which are known to be important for seed germinability, was determined by semi-quantitative RT-PCR in embryos during the period of seed germination. The expression of OsGA20ox3, OsGA20ox4 and OsGA3ox1 was not detected in this experiment.
The qLTG-3-1 gene clearly induced the expression of OsGA20ox1, OsGA20ox2, OsGA3ox2 and Ramy 1A, all of which have expression patterns similar to that of qLTG-3-1 (
Expression delays in all the tested genes were examined in Hayamasari. Italica Livorno and NILHYqLTG-3-1 showed the same expression patterns, which suggests that the expression of these genes in GA biosynthesis and Ramy 1A is induced by qLTG-3-1. In addition, the expression of eight genes for GA biosynthesis and amylase was determined (Table 5,
In this example, the overexpression of qLTG-3-1 was carried out. To create overexpressing plants, a construct of the 35S promoter which drives qLTG-3-1 was created. PCR product amplified using the primers Ano13-LA5U and Ano13-LA5L (Table 4) was digested with BamHI and SacI. These fragments were cloned to the Sad site of the pPZP2Ha3 Ti-Plasmid vector (Fuse et al, 2001).
An Agrobacterium-mediated transformant was used to transform Hayamasari (Toki, 1997; Toki et al., 2006). Plants regenerated from hygromycin-resistant calluses (T0 plants) were grown in an isolated greenhouse. Self-fertile plants of each T0 plant (T1) were grown. The T1 transformants were screened by PCR for transgenes and cultivated to obtain T2 plants, which were collected in order to carry out germination experiments.
qTLG-3-1 has a tissue-specific expression. In order to examine the effects of expression pattern, tissue specificity and amount on germination in the plant and on regulation of the stress response, overexpressed plants in which the qLTG-3-1 is driven by the 35S promoter were created. In transgenic plants, qLTG-3-1 was strongly expressed in the leaves and panicles; however, expression was not detected in nontransgenic plants (
In this example, the DNA polymorphism in the qLTG-3-1 gene within cultivated rices and wild varieties was examined. The seeds of a core collection of cultivated rice (O. sativa) containing 62 varieties (Kojima et al., 2005) were acquired from the National Institute of Agrobiological Sciences. This collection was composed of three groups, Groups A, B and C, which correspond respectively to Japonica, Aus and Indica from a wide region (Kojima et al., 2005; Garris et al., 2005).
Rice seed of the AA genome wild species W0106 (O. rufipogon), W0652 (O. barthii), W1169 (O. glumaepatula), W1413 (O. longistaminata) and W1508 (O. longistaminata) were acquired from the National Institute of Genetics of the Research Organization of Information and Systems. The total DNA was isolated from young leaves using the CTAB method described by Fujino et al. (2004).
The qLTG-3-1 gene was amplified using primers (Table 4), then directly sequenced using cycle sequencing with a Big Dye Terminator (Applied Biosystems). Sequencing was carried out with a Prism 3700 automated sequencer (Applied Biosystems). Alignment of the DNA sequences was carried out using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), following which the sequences were visually confirmed. All polymorphisms were rechecked from chromatograms while paying particular attention to low-frequency polymorphisms.
Heterozygosity was not observed in the sequences found by the inventors. A 1,784-bp gene in qLTG-3-1 that includes a 933-bp upstream region containing 5′ and 3′UTR and a 296-bp downstream region, excluding TA repeats in the 50-bp region from base pairs 433 to 384, was sequenced in the cultivated rices and the wild varieties. The minimum range (haplotype) exhibiting a unique DNA sequence or an allele separated by a mutation step was constructed by the computer program TCS (Crandall et al., 2000) using the parsimony method in statistics.
All 1,784 nucleotides of the qLTG-3-1 gene were sequenced. Compared with the Italica Livorno sequence as a functional allele, 32 mutations—including insertions, deletions and substitutions—were detected (Table 6). Based on these mutations, ten different haplotypes were found among the 62 varieties in the rice core collection (Table 7; DNA sequences,
1)Kojima et al. (2005)
Two deletions, one insertion and one non-synonymous substitution arose in the gene coding region. All the deletions and insertions in the signal region arose as 3n by in-frame mutations. One non-synonymous substitution was detected in Nipponbare alone. Haplotypes 9 and 10 appear to have been obtained from intragenic recombinations between Haplogroups I and II.
A haplotype network was constructed from the gene mutations in the entire qLTG-3-1 gene. Because two haplotypes arose from intragenic recombinations, these were not included in the network. The network was composed of the two Haplogroups I and II (
These haplogroups were mutually segregated in 18 mutation steps. Haplogroup I containing five haplotypes was composed of 31 varieties, most of which were from Groups A and B of the core collection of rice. Haplogroup II containing three haplotypes was composed of 25 varieties, most of which were from Groups B and C.
The qLTG-3-1 sequences of relative rices in the AA genome were compared with the qLTG-3-1 functional allele (Haplotype I) of Italica Livorno. Numerous nucleotide changes, including insertions, deletions and substitutions, were detected. However, these were not detected in cultivated rices. Most of these nucleotide changes arose in O. longistaminata (W1413 and W1508), which clearly differed from the other lines.
Thirteen synonymous substitutions, 6 deletions and 11 insertions were detected in the gene coding region. All the deletions and insertions arose in-frame within the GRP region (
As described above, the present invention relates to a gene for low temperature germinability in rice, and a method for its use. Through this invention, there can be provided both a gene for a low temperature germinability isolated from the rice line Italica Livorno, and the base sequence of the gene. The present invention also makes it possible to provide a technique for utilizing the foregoing gene that employs the functions of the above gene alone for the low temperature germinability, e.g., the germinability-improving properties under low temperature, salt and osmotic pressure stresses; to provide a transgenic plant in which the above gene has been transformed to improve the low temperature germinability; and to differentiate between the levels of low temperature germinability in cultivated varieties by identifying gene mutations based on the sequence of the gene for the low temperature germinability. In the past, from QTL analysis, the high level of low temperature germinability recognized in Italica Livorno was thought to be a quantitative trait in which a plurality of genes participated. The present invention is useful in that it provides an isolated gene alone for the low temperature germinability as a substance, as well as the base sequence thereof, and also provides a technique for the use of the gene.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007 233461 | Sep 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/066110 | 9/5/2008 | WO | 00 | 7/6/2010 |
