JP2010154848A - Uvi4-like gene deleted plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To form an unreduced gamete having reproductive ability in both of maleness (pollen) and femaleness (ovule) by mutation of a single gene, and thereby to provide a plant capable of preparing a progeny seed having about two times genome amount of the parent plant by self-propagation. <P>SOLUTION: There is provided the plant decreased or deleted in function of a UVI4-like gene or another homologue gene having a specific cDNA base sequence, forming an unreduced gamete having reproductive ability in both of maleness (pollen) and femaleness (ovule) to obtain a progeny seed having about two times genome amount by self-propagation. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention forms a non-reducing gamete that is fertile on both the male side (pollen) and the female side (egg) by reducing or deleting the function of the UVI4-like gene. The present invention relates to a plant capable of obtaining a progeny seed having a double genome amount.

  Recent genome analysis revealed that multiple genome-wide doubling occurred in the evolution of angiosperms (Non-patent Document 1). Even Arabidopsis, which has the smallest genome size in plants, is believed to have experienced at least three whole genome doublings (Non-Patent Document 2; Non-Patent Document 3). Thus, it is believed that many diploid plants that exist today are actually polyploid.

  From previous studies, the formation of non-reducing gametes has played a major role in the formation of polyploids, and polyploids have been formed via triploids formed by fertilization with non-reducing gametes. (Non-patent document 4; Non-patent document 5). The frequency of formation of non-reduced gametes is generally very low, but this phenomenon has been confirmed in many plant species (Non-patent Document 6; Non-patent Document 7).

  Studies of several meiotic mutants in potato have shown that the formation of non-reducing gametes can occur under the control of a single recessive factor, but the gene has not been isolated (8). Non-patent document 9). In Arabidopsis, it has been reported that non-reducing female gametes capable of fertilization are formed by mutation of one gene (Non-patent Document 10). Although it is reported that 80% of the progeny progeny plants of this mutant strain were triploid, the frequency of formation of non-reduced gametes is low, and the number of seeds obtained is extremely low at 10 or less per individual. there were.

  In addition to the pathway through which triploids yield higher polyploids than tetraploids, tetraploids may be obtained directly by fertilization between non-reducing female and male gametes, Because of the generally low frequency of non-reduced gametes that have been identified to date and the absence of correlation between the formation of non-reduced gametes in female and male gametes, there is no correlation between non-reduced female and male gametes. It was thought that the route by which the tetraploid was directly obtained by fertilization of was not likely to occur. In addition, the gene that leads to the formation of non-reducing gametes that are fertile on both the male side (pollen) and the female side (egg), as well as the phenomenon that one gene mutation directly produces a tetraploid progeny plant. Not reported.

In addition, the UVI4-like protein whose sequence is described in SEQ ID NO: 2 is a UVI4 (UV-B insensitive 4, At2g42260) protein (non-inverted) that has been reported to increase nuclear doubling in leaves by mutation. Patent Document 11 and Patent Document 1) have high similarity in amino acid sequence, but have not been proven so far, and there is no estimation of functions related to features other than the feature that mutations increase nuclear doubling. Is possible.
JP 2007-228922 Comai (2005) Nature Rev. Genet. 6, 836-846. Vision et al. (2000) Science290, 2114-2117. Adams & Wendel (2005) Curr. Opin. Plant. Biol. 8, 135-141. Ramsey & Schemske (1998) Ann. Rev. Eco. Syst. 29. 467-501. Ramsey & Schemske (2002) Ann. Rev. Eco. Syst. 33. 589-639. Veilleux (1985) Plant Breed. Rev. 3, 253-288. Bretagnolle & Thompson (1995) New Phytologist 129, 1-11. Carputo et al. (2003) Genetics163, 287-294. Peloquin et al. (1999) Genetics 153, 1493-1499. Ravi et al. (2008) Nature 451, 1121-1125. Hase et al. (2006) Plant J. 46, 317-326.

  As known by the term “polyploid breeding”, increasing the ploidy of a plant can lead to an increase in yield.

  The present invention identifies a novel gene in which non-reducing gametes capable of reproduction are formed on both the male side (pollen) and the female side (egg) by reducing or deleting its function in plants. Mutations form fertile non-reduced gametes on both the male side (pollen) and the female side (egg), resulting in progeny seeds that have about twice the genome amount of the parent plant by selfing It is an object to provide plants that can be used.

  The present inventors analyzed the characteristics of the Arabidopsis UVI4-like (UV-B insensitive 4-similar, At3g57860) gene disruption strain. As a result, in plants where the function of UVI4-like protein was reduced or deleted, It was found that fertile non-reducing gametes were formed on both the male (pollen) side and the female (egg) side. Furthermore, it has been found that progeny seeds having twice the genome amount of the plant can be obtained by self-propagation of the plant, and the present invention has been completed.

  That is, the present invention provides a plant in which the function of the UVI4-like gene or other species homolog gene whose cDNA base sequence is described in SEQ ID NO: 1 is reduced or deleted based on the novel findings as described above.

  In a preferred embodiment, the plant of the present invention forms a reproductive non-reducing gamete on both the male side (pollen) and the female side (egg). In a more preferred embodiment, the plant of the present invention can obtain progeny seed having about twice the genome amount by self-propagation.

  Furthermore, another preferred embodiment of the present invention is a cruciferous plant, and more particularly, Arabidopsis thaliana.

  According to the present invention, it is possible to provide a plant that forms a non-reducing gamete having fertility on both the male side (pollen) and the female side (egg). In addition, progeny seeds having twice the genome amount of the plant can be produced by self-propagating the plant.

Amino acid sequence alignment of At3g57860 and putative homolog. The same amino acid as that of At3g57860 is shown in white. Two 鏃 marks indicate the insertion positions of the Ds transposon in the pst11867 and pst15307 lines. Sequence alignment was prepared using Clustal W program v1.83 (http://www.ddbj.nig.ac.jp/). Schematic diagram and nucleotide sequence of Ds transposon insertion site. The region encoding the protein of At3g57860 gene is indicated by a gray square. Triangle indicates Ds transposon. The arrow indicates the primer used to determine the insertion position. The length and genome sequence of the PCR fragment amplified by the primers (Ds5-2a, Ds5-3, Ds3-2a and Ds3-4) located at the end of the Ds transposon and the primers designed on the genomic DNA (X and Y) are , Shows that Ds transposons were inserted in different directions in both strains. The sequence of the PCR fragment showed that in the pst11867 strain, a Ds transposon was inserted at the +223 nucleotide position indicated by the arrow from the start codon and was accompanied by a 14-bp target replication indicated by the boxed sequence. . On the other hand, the pst15307 line was inserted at +270 and was accompanied by a 7-bp replication. The underlined sequence indicates the sequence at the end of the Ds transposon. The highlighted sequence shows the base sequence of the footprint generated by retransfer of the Ds transposon. Formation of large pollen by bimolecular formation. (a) Wild type pollen. (b, c) Heterozygous normal size pollen. (d) Wild-type four molecules. (e) Large pollen formed of heterozygote. (f, g) Large pollen formed from homozygote. (h) Bimolecule observed in homozygote. (i, j) Pollen in pollen chamber of homozygote (i) and wild type (j) plant body. Mature pollen was stained with Alexander stain (a, b, e, f, i, j) or Hoechst 33342 (c, g). Four molecules (d) and two molecules (h) were stained with toluidine blue. The scale bar has a length of 30 μm (a, b, d−f, h), 20 μm (c, g), and 100 μm (i, j). The size of pollen formed from a self-propagating progeny plant of a double-ploid heterozygote of a single pst11867 line extracted at random. The data shows the mean value ± standard deviation of the major axis of the pollen obtained from each individual. Comparison of pollen in the tub. The anther just before flowering was stained with carmine acetate. The homozygote (center) forms larger non-reducing pollen than the wild type (left) pollen. A tetraploid homozygote (Ds / Ds / Ds / Ds) (right) obtained by self-breeding of individuals that form large pollen is a large pollen that is estimated to contain tetraploid DNA. Form. Seed formation with increased ploidy due to UVI4-like gene deficiency. (a) Seeds in the sheath of wild type (left), homozygote (middle) and heterozygote (right) forming large pollen. (b, c) Seeds obtained from wild type (b) and homozygote (c). The scale bar is 2mm for (a) and 1mm for (b, c). Male meiosis in wild type and homozygotes. Chromosome extension specimens of wild type (a-d, i, j) and homozygote (e-h, k) are shown. (a, e) pachytene period. (b, f) Diakinesis period. (c, g) Middle meiosis. (d, h) From the end of the first meiosis to the second meiotic prophase. (i) Second meiotic metaphase. (j) End of second meiosis, (k) End of first meiosis. The scale bar has a length of 10 μm and is common to all figures. Distribution of male meiotic cells in wild type and homozygotes. The ratio to the total number of cells observed is shown. For observation, five inflorescences were used for each of the wild type and homozygote.

  The present invention is a plant in which the function of the UVI4-like gene or other species homologue gene is reduced or deleted. The term “reduced or deleted gene function” is a decrease or deletion of transcription of the gene. Or reduced translation of the gene into protein or protein deletion.

  Such a decrease in function or trait of a gene can be achieved by inserting a transposon into a target gene (UVI4-like gene or other species homolog gene), as shown in the Examples below. In addition, methods used in this technical field for functional reduction or deletion of plant genes can be appropriately used. For example, a method that introduces a DNA encoding an antisense RNA complementary to a target gene into a plant body, or a DNA that encodes a ribozyme (hammerhead ribozyme or hairpin ribozyme) that specifically cleaves the target gene mRNA. And a method using DNA encoding a complementary double-stranded RNA (dsRNA) to the mRNA of the target gene. Alternatively, an RNA silencing method by introducing a virus vector incorporating a target gene into a plant can be used. Furthermore, a gene knockout method based on a gene targeting method targeting a target gene in genomic DNA can also be used. That is, the UVI4-like gene is known to be located in the 21437291-21439230 base sequence of Arabidopsis thaliana chromosome 3 (GenBank / NC_003074), and gene targeting method using a homologous recombination vector prepared based on this genomic sequence information By knocking out the gene, the function of the target gene can be deleted.

  In the present invention, “UVI4-like gene or other homologous gene” refers to a UVI4-like gene of Arabidopsis thaliana or a UVI4-like homologous gene of a plant other than Arabidopsis thaliana (a structure and function homologous to the Arabidopsis UVI4-like gene). Gene). Plants other than Arabidopsis thaliana can include, but are not limited to, poplar, grapes, rice (FIG. 1) and the like, which are known to have homology to UVI4-like genes. For example, UVI4-like homologous genes can be isolated from useful plants of Brassicaceae (cabbage, radish, etc.) to which Arabidopsis belongs, and the functions of UVI4-like homologous genes of these cruciferous plants can be reduced or deleted. To isolate a UVI4-like homolog gene, for example, a PCR method using a primer oligonucleotide prepared based on the sequence information (SEQ ID NO: 1) of the UVI4-like gene, or a sequence based on the sequence information. It can be performed according to a method for identifying a homologous gene in the art, such as a method for screening a cDNA library of a specific plant using a probe oligonucleotide.

  Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.

(1) Method
(1-1) Plant material and growth conditions Arabidopsis thaliana Ds transposon taglines (strain names pst11867 and pst15307) were provided by the RIKEN BioResource Center. These strains were created using the Arabidopsis No-0 ecotype as a background. Plants are grown in commercially available culture soil (MetroMix350, Scotts-Sierra Horticultural Products Company, Marysville, OH, USA) in a growth room equipped with a white fluorescent lamp of 3,000-4,000 lux at 23 ° C for 16 hours. I went there.
(1-2) Genotyping by PCR Genomic DNA is Kurabo Industries's PI-50α automatic DNA extraction system, Qiagen's DNeasy Plant Mini Kit, Sigma's Extract-N-Amp Plant PCR Kits or DNA extraction solution (200 mM Tris- Extraction was performed using HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA and 0.5% SDS. The genotype survey is based on the combination of primer sequences specific for the UVI4-like gene indicated by X and Y in FIG.
X: 5'-TAGACGTTAGGGTGGGGTTTAC-3 '(SEQ ID NO: 3) and
Y: 5'-CAACAGGAAAACACAAAAGAGC-3 '(SEQ ID NO: 4),
In addition, Ds5-2a, Ds5-3, Ds3-2a and Ds3-4 primer sequences specific for the Ds transposon (Kuromori et al. (2004) Plant J. 37, 897-905) were used. For detection of Ac element, a combination of primers specific to the base sequence of Ac element
Ac2656: 5'-TAAAGCCGAGGAGTGGAAGA-3 '(SEQ ID NO: 5) and
Ac3244: 5′-TCCCCTCCACCATGATAAAA-3 ′ (SEQ ID NO: 6) was used.
(1-3) Microscopic observation Mature pollen was stained with Alexander staining solution (Alexander et al. (1969) Stain Technology 44, 117-122). The pollen in the cocoon chamber was observed by staining the cocoon just before opening with a 1% carmine acetate solution or an Alexander staining solution overnight and then gently crushing it on a slide glass. The pollen diameter was measured by staining mature pollen with a 1% carmine acetate solution. Pollen diameter was measured using the software ImageJ 1.37v. More than 50 grains of pollen were measured for each plant. Nucleus size was observed by staining mature pollen with a 0.1% solution of Hochest33342. Tetramolecular and bimolecular observations were made by placing fresh strawberries in a 0.02% solution of toluidine blue and using a scalpel to remove the contents onto a glass slide. Chromosome extension specimens of male meiotic cells were prepared by the method described in the literature (Ross et al. 1996) and stained with 10 μl of 4 ′, 6-diamidino-2-phenylindole (1 μg / ml).
(1-4) Ploidy analysis Using a young rosette leaf of a 2-3 week old plant body, it was measured by the method described in the literature (Haseet al. (2006) Plant J. 46, 317-326). The amount of DNA at the lowest peak position was measured using Partec software DPAC. The young leaves of soybean with about 8.4 times the nuclear DNA content of Arabidopsis thaliana were measured simultaneously as an internal standard.
(2) Results and discussion
In order to investigate the function of the UVI4-like gene, two strains (pst11867 and pst15307) were obtained in which the UVI4-like gene was disrupted by insertion of a Ds transposon. From the results of PCR and nucleotide sequence analysis, it was found that the Ds transposon was inserted at position +223 for pst11867 and position +270 for pst15307 when the translation start point of the nucleotide sequence of the UVI4-like gene was +1 (Fig. 2). From these insertion positions, the two strains of disrupted strains are presumed to be loss-of-function strains. None of the eleven pst11867 strains examined by PCR-based genotyping have homozygous knockout alleles (UVI4-like alleles that have lost function due to transposon insertion). it was thought. Furthermore, since the seed sizes were not uniform, the ploidy was investigated by measuring the amount of nuclear DNA. As a result, 5 of the 11 individuals were considered to be tetraploid. From these results, it was considered that the 11 pst11867 lines examined were 2 diploid wild types, 4 diploid heterozygotes, and 5 tetraploid heterozygotes. This result suggests that ploidy is increased by disruption of the UVI4-like gene. Similar results were also observed in the pst15307 line, suggesting that the observed phenomenon was due to UVI4-like gene disruption.

  The 204 inbred progeny genotypes obtained from randomly selected pst11867 strain diploid knockout allele heterozygotes were 47 wild types, 141 heterozygotes and 16 homozygotes. It was considered to be a joined body. Of the 204 individuals, the ploidy of a total of 52 individuals including 16 wild-types, 27 heterozygotes and 9 homozygotes was measured. As a result, all plants were diploid. Here we found that all homozygotes as well as some heterozygotes (25 of 141 individuals) formed larger than normal pollens (Table 1 and FIGS. 3e, f). The large pollen had three nuclei typical of Arabidopsis pollen, two brightly stained sperm nuclei and one faintly stained vegetative nucleus (FIG. 3g). However, the fact that the size of the nucleus is clearly larger than that of the wild type shown in FIG. 3c indicates a high ploidy. Heterozygote is divided into either an individual that forms large pollen or an individual that forms pollen of the same size as wild type, and an individual that forms both large pollen and pollen of the same size as wild type It was not confirmed (FIG. 4). The long diameter of the pollen formed by the homozygote was 29.9 ± 1.1 μm, whereas the long diameter of the wild type pollen was 25.0 ± 1.0 μm (FIG. 4). The wild type cocoon contained a total of 420 to 440 pollen, but it decreased to 240 to 280 in the individuals forming large pollen. In individuals that formed large pollen, the size of the pollen was almost uniform, and almost no dysplastic pollen was observed (FIGS. 3i and 5).

  In homozygotes and heterozygotes that formed large pollen, transcripts of the UVI4-like gene were not detected by RT-PCR. The results of previous studies suggested that the pst11867 strain may have an Ac element that translocates transposons. A PCR product specific for the Ac element was confirmed from the heterozygote forming large pollen, and the footprint base sequence left after the relocation of the Ds transposon was confirmed at the UVI4-like locus. (FIG. 2). Since genotyping by PCR is based on the length of amplified DNA, it is possible to distinguish between normal UVI4-like loci and UVI4-like loci that have lost function due to retransfer of Ds transposons. Can not. These results suggest that heterozygotes that form large pollen have both a locus into which the Ds transposon has been inserted and a locus that has lost its function due to retransfer of the Ds transposon. Furthermore, a complementary test between the pst15307 strain and the pst11867 strain that does not contain an Ac element showed that both strains had mutations in the same gene, indicating that the phenotype that forms large pollen is a defect in the UVI4-like locus. It was shown to occur by activation. These results indicate that Arabidopsis diploids exhibit a large pollen-forming phenotype only when both UVI4-like loci are inactivated, ie, only in recessive homozygotes. .

  Viable seeds were obtained by self-propagation of individuals that formed large pollen, which were clearly larger than the seeds obtained in the wild type (FIG. 6). As a result of the ploidy investigation, 19 out of 20 individuals obtained from homozygotes (= 95%) were tetraploid, and the remaining one was considered to be triploid (Table 2). Individuals estimated to be tetraploid were also obtained by self-breeding of heterozygotes that formed large pollen (63 of 73 individuals = 83%) (Table 2). These results indicate that fertile non-reducing gametes are formed on both the male (pollen) side and the female (egg) side by disruption of the UVI4-like gene. In addition, since a small number of triploids were obtained, both normal haploid gametes and non-reduced gametes are formed on either the male (pollen) side or the female (egg) side. Is suggested.

  In normal Arabidopsis pollen formation, four molecules are formed by meiosis (FIG. 3d). In contrast, only two molecules were observed at the same time in individuals that formed large pollen (n = 281) (FIG. 3h). Tetramolecules and other abnormal meiotic cells were not observed. This result shows that disruption of the UVI4-like gene causes meiotic abnormalities in male (pollen) gametogenesis, and non-reduced pollen is formed with a frequency close to 100%.

  Furthermore, in order to investigate abnormalities of meiosis, forward and reverse mating with the wild type was performed. F1 plants obtained by crossing a homozygote with a wild type as a pollen parent were considered to be triploid in all 35 individuals. On the other hand, when the homozygote was crossed with the wild type as a seed parent, 15 of the 23 F1 plants examined were triploid and 8 were diploid. These results show again that non-reducing gametes that are fertile are formed on both the male side (pollen) and the female side (egg). Furthermore, on the male side (pollen), the frequency of formation of non-reducing gametes is almost 100%, while on the female side (eggs) both normal haploid gametes and non-reducing gametes are formed. It has been shown. As a control experiment, when a heterozygote that forms pollen of the same size as the wild type is crossed with the wild type, all F1 plants are doubled regardless of whether they are used as the pollen parent or the seed parent. Yes, it was confirmed that the genotype segregated wild type and heterozygote 1: 1.

  The tetraploid homozygote formed giant pollen with a diameter of about 37 μm. An individual estimated to be octaploid was obtained by self-breeding of this individual. However, the octaploid was sterile and no progeny was obtained.

  In order to investigate further meiotic abnormalities, we observed chromosome dynamics in male meiosis. Since the dynamics of chromosomes in the wild type are described in detail in the literature (Ma (2006) The Arabidopsis Book, June 6), etc., they will be briefly described here. First, in the leptotene phase of prophase I, a slightly condensed thin linear chromosome is observed. Thereafter, pairing of homologous chromosomes begins in the zygotene phase. In the pachytene phase, homologous chromosomes are perfectly paired, and a thick filamentous chromosome is observed (FIG. 7a). The pairing between homologous chromosomes begins to dissociate in the diplotene phase, the homologous chromosomes further condense, and a structure called chiasma in which the chromosomes crossed in the diakinesis phase is observed (FIG. 7b). Five pairs of homologous chromosomes, called divalent chromosomes, are arranged in the equatorial plane at the first metaphase I (Fig. 7c). Homologous chromosomes are separated into two poles at the first meiotic anaphase (anaphase I), and the chromosomes are slightly decondensed from the first meiotic terminal (telophase I) to the second meiotic prophase (prophase II) (FIG. 7d). Two sets of five chromosomes are arranged on different equator planes in the second metaphase II (metaphase II) (Fig. 7i), and the second chromosome segregation takes place in the second meiotic late phase (anaphase II). Is called. The second meiotic phase (telophase II) is the last stage of meiosis, and four haploid cells called tetramolecules are formed (FIG. 7j). In the homozygote of the knockout allele of the UVI4-like gene, no significant difference from the wild type was found in the first meiosis. That is, pairing between homologous chromosomes (FIG. 7e) and formation of chiasma (FIG. 7f) were considered normal. In addition, five pairs of divalent chromosomes were arranged on the equator plane (FIG. 7g) and then separated into two poles (FIG. 7h). However, no chromosome image corresponding to the second meiosis was observed. From the end of the first meiosis to the second meiotic prophase, the chromosomes are somewhat decondensed in the wild type (Fig. 7d), but such decondensed chromosomes are rare in homozygotes, many of which are shown in Fig. 7k. He had condensed chromosomes. Since this condensed chromosome is similar to the chromosome appearance at the end of the second meiosis in the wild type (Fig. 7j), homozygotes may end meiosis without performing the second meiosis. It was suggested.

  In order to investigate the progression of meiosis, the proportion of cells belonging to each stage of meiosis was investigated (FIG. 8). In the wild type, the proportion of cells in the zygotene phase and the pachytene phase was large, and the proportion of cells in the second meiotic phase was also large. The proportion of cells in the middle and late stages of the first and second meiosis was low (less than 1% of the total). In homozygotes for the knockout allele of the UVI4-like gene, the distribution in the first prophase I (prophase I) was similar to that of the wild type. However, the proportion of cells in the first meiotic metaphase (4.1% of the total) was significantly higher than that of the wild-type stage (0.5%). No second meiotic cells were observed. These results suggest that homozygotes delay the progression of meiosis from the first metaphase to late meiosis.

  It is known that a protein complex called Anaphase Promoting Complex (APC) plays an important role in the transition from metaphase to late phase of cell division (Eloy et al. (2006) Cell Cycle 5, 1957-1965). . APCs target fission cyclin and degrade it, resulting in inactivation of the fission cyclin-cyclin-dependent kinase complex, thereby promoting late transition. Appropriate control of mitotic cyclin-cyclin-dependent kinase activity is also considered important for the transition from first meiosis to second meiosis (Pesin and Orr-Weaver (2008) Annu. Rev. Cell Dev. Biol. 24, 475-499). From the above, it is suggested that the APC activity may be affected by the mutation of UVI4-like.

Claims (6)

A plant in which the function of the UVI4-like gene or other species homologue gene whose cDNA base sequence is described in SEQ ID NO: 1 is reduced or deleted. 2. A plant according to claim 1 which forms fertile non-reducing gametes on both the male side (pollen) and the female side (egg). The plant according to claim 1, wherein non-reducing gametes on the male side are formed with a frequency close to 100%. The plant according to claim 1, wherein progeny seeds having about twice the genome amount are obtained by self-propagation. The plant according to claim 1, which is a cruciferous plant. The plant according to claim 5, which is an Arabidopsis thaliana.