JP2006006122A - Method for selecting transgenic plant using ethanol-resistant gene and transforming vector therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently selecting a transgenic plant by solving difficulty of discrimination between a transgenic plant and a non-transgenic plant in the case of selecting a transgenic plant by using a medicine-resistant gene. <P>SOLUTION: The method for selecting a transgenic plant comprises a process for transducing a recombinant DNA containing a GEK1 gene to an ethanol-sensitive plant, a process for cultivating the plant to collect seeds and a process for sowing the seeds in an ethanol-containing medium for plant growth to germinate a transgenic plant. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for selecting a transformed plant into which a recombinant DNA has been introduced and a vector for transforming the plant, and more particularly to a method and a vector for selecting a transformed plant using ethanol resistance as an index.

  In recent years, varieties have been improved by introducing exogenous genes into plants using gene recombination techniques and imparting superior traits such as resistance to environmental stress. In order to produce such a transformed plant, it is necessary to select a cell or an individual in which the target gene (DNA fragment) is inserted on the genome. At present, microorganism-derived antibiotic resistance genes such as hygromycin resistance gene (see Non-Patent Document 1), kanamycin resistance gene, chloramphenicol resistance gene (see Non-Patent Document 2) are mainly used. In order to select transformants carrying these marker genes, seeds are generally sown on an agar medium containing drugs such as antibiotics and grown for several weeks. Individuals that do not have a drug resistance gene will germinate but gradually die out as growth is inhibited. On the other hand, individuals with drug resistance genes grow better and are affected by drugs, but do not die.

Arabidopsis is a small weed of the Brassicaceae family, and its genome size is about 1.3 × 10 ⁸ base pairs, which is the smallest among higher plants, and the time required for one generation is as short as about 2 months. As widely used worldwide. The entire base sequence of the genome has already been decoded by an international project. The base sequence of one gene GEK1 existing in the genome of this plant (for example, see Non-Patent Document 3) is already known, but the function of its putative gene product and the physiological role in the plant have not yet been elucidated. Not.

Gene, 25-25, 179-188, 1983 Nucleic Acids Research, Vol. 11, No. 13, pp. 6981-6998, 1985 GenBank Accession No. AB091252 [gi: 28812212], February 14, 2004

  In the above-mentioned prior art, the problem when selecting a transformed plant using a drug resistance gene as a marker is first to distinguish between a resistant plant (a plant having a resistant gene) and a non-resistant plant (a plant having no resistant gene). To do this, the plant must be grown for at least 7 days. In some cases, selection requires a discriminating ability that has been trained. Second, when growth is delayed by a target gene introduced together with a drug resistance gene, or when greening is suppressed, it becomes difficult to distinguish between a transformed plant and a non-transformed plant. Third, the drug resistance gene is originally a gene of another biological species, and may not be suitable for consumers in producing a genetically modified crop.

  In order to solve the above problems, the present inventors have found that one gene GEK1 originally possessed by higher plants is involved in ethanol tolerance of plants, and that this GEK1 gene is used to characterize ethanol tolerance as an index. The present invention was completed by finding that a converted plant can be selected efficiently. For example, Arabidopsis thaliana Heinth L., a plant in which this gene product has stopped functioning, is about 100 times more sensitive to ethanol than the wild type, and a medium containing ethanol at a low concentration of 0.003%. Germination is also inhibited above. However, this disrupted strain has no other phenotype and grows normally to form seeds. When the GEK1 gene is introduced into this mutant plant by an Agrobacterium-mediated method, ethanol resistance similar to that of the wild strain is restored, and germination can be achieved even in the presence of 1% ethanol. The present invention is a system in which a transformed plant can be selected at the time of germination by using a plant having a mutation in the Arabidopsis GEK1 gene or a GEK1-like gene of another plant and a GEK1 gene incorporated in a vector. Is to provide.

  That is, the method for selecting a transformed plant according to one aspect of the present invention includes a step of introducing a recombinant DNA containing a GEK1 gene into an ethanol-sensitive plant, a step of growing the plant and collecting seeds, and ethanol. And sowing the seed in a plant growth medium to germinate the transformed plant. The ethanol-sensitive plant preferably has a mutation in the genomic endogenous GEK1 gene. The plant is a monocotyledonous plant or a dicotyledonous plant, and is preferably, for example, sunflower, sunflower, potato, cotton, soybean, tomato, rice, leek, corn, barley, or wheat.

  A plant transformation vector according to another aspect of the present invention is a DNA encoding the amino acid sequence shown in SEQ ID NO: 2 or has at least 70% homology with the amino acid sequence, and the plant has ethanol resistance. It contains DNA encoding the protein to be imparted. The vector preferably further contains a target gene to be expressed in the plant.

  According to the method of the present invention, selection with or without germination can be performed using a plant-derived gene. Selection based on the presence or absence of germination is very easy to distinguish visually, and the selection by an image recognition apparatus can be automated. Further, since it is not necessary to grow all the individuals, more individuals can be selected per unit area. Furthermore, since the GEK1 gene used in the method of the present invention is a gene originally possessed by the host plant, the drug resistance gene of a heterologous organism can penetrate (transfer) from the transformed plant to the wild type plant by gene introgression. It is also considered to be highly safe for application to genetically modified crops.

  BEST MODE FOR CARRYING OUT THE INVENTION A best mode of a method for selecting transformed plants according to the present invention (referred to as “selection method of the present invention”) and a recombinant vector for transformation used in the method will be described in detail with reference to the drawings.

<Gene conferring ethanol tolerance on plants>
In the present invention, “GEK1 gene” means DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence and at least 50%, preferably 70%, 80%, 85%, 90%, 97 %, A gene encoding a protein having a homology of 98% or more and imparting ethanol tolerance to the plant. The degree of protein homology can be expressed as a percentage of identity when the amino acid sequences of two proteins are properly aligned, and the rate of occurrence of exact matches between the sequences can be expressed as means. Appropriate alignment between sequences for identity comparison can be determined using various algorithms, such as the BLAST algorithm (Altschul SF J Mol Biol 1990 Oct 5; 215 (3): 403-10). This GEK1 gene was first discovered by the present inventors in Arabidopsis thaliana and exists in a locus named GEKO1 (refers to a person who cannot drink alcohol in Japanese. ”). In the present specification, the expression “GEK1” means a wild type (dominant) gene or protein that can confer ethanol tolerance to a plant, and the expression “gek1” suddenly indicates the gene. It means a mutant (recessive) gene or protein whose function is suppressed (attenuated) or impaired by mutation, for example, deletion, addition, or substitution of a base sequence.

  The protein deduced from the genomic GEK1 gene derived from Arabidopsis thaliana consists of 361 amino acids. Among these, the cDNA sequence encoding 317 amino acids starting from the 45th methionine residue from the N-terminus is shown in SEQ ID NO: 1. . It has been clarified by analysis using a database that the GEK1 gene exists in the genomes of various plant species regardless of dicotyledonous or monocotyledonous plants. To date, it seems to encode a similar protein (more than 70% of comparable amino acid sequences) to GEK1 protein (SEQ ID NO: 2) derived from Arabidopsis thaliana among cDNAs that have been partially sequenced Found in sunflower, sunflower, potato, cotton, soybean, tomato, rice, leek, corn, barley and wheat. Since the similarity of amino acid sequences is high, these gene products are considered to have a function similar to that of GEK1 protein derived from Arabidopsis thaliana. Therefore, it is considered that the same selection method as that of Arabidopsis thaliana can be used for the above-mentioned plant by the method of the present invention by isolating the mutant of the GEK1 gene. As an example, sequence homology with tomato (estimated from two tomato EST base sequences of BE432622 and AW650002) and a similar gene of rice (GenBank accession BAD03648) is shown (FIG. 1). As shown in FIG. 1, the sequence after the 45th methionine residue of GEK1 protein shows high homology among these plants. Further, it has been experimentally confirmed that a protein expressed with the 45th methionine as the N-terminus has the same activity as a protein starting from the 1st methionine. On the other hand, there are no GEK1-like genes in animals and microorganisms whose whole genome sequences have been decoded so far, such as humans and mice, E. coli and yeast. Note that the structure of the GEK1 protein does not contain any known motif.

  Further, in each of the above proteins, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, and imparting ethanol tolerance to plants is also included in the GEK1 protein. For example, those skilled in the art are able to produce a biologically equivalent protein by changing several amino acids to the amino acid sequence of the protein shown in SEQ ID NO: 2 without substantially changing the biological function of the protein. Well known. In one aspect of the invention, the GEK1 protein may comprise a protein that differs from the wild type GEK1 protein by conservative amino acid substitutions. The term “conservative amino acid substitution” refers to the replacement of one amino acid with another amino acid at a particular position in the protein. Such a substitution is a substitution with a similar amino acid, and is performed based on the relative similarity of the side chain substituents of the amino acid, such as size, charge, hydrophobicity, hydrophilicity, and the like.

  Although the biological role of the GEK1 protein has not yet been clarified, the sensitivity of the mutant lacking the GEK1 gene is ethanol-specific, suggesting an association with ethanol metabolites. Ethanol in the medium is thought to be converted to acetaldehyde by aldehyde dehydrogenase (ADH). Thus, when the gekl phenotype and ADH activity were examined, it was found that ADH activity is essential for ethanol sensitivity due to the deletion of the GEK1 gene. That is, in the ADH-deficient strain, the ethanol sensitivity due to the GEK1 mutation disappears, and therefore the gek1 mutant strain is considered to be highly ethanol-sensitive due to the toxicity due to acetaldehyde produced by ethanol metabolism.

<Production of recombinant vector>
In the present invention, the vector used for transformation of a plant or plant cell is not particularly limited as long as the inserted gene can be expressed in the cell. For example, a vector having a promoter (eg, cauliflower mosaic virus 35S promoter or rice actin promoter) for constitutive gene expression in plant cells, or a promoter that is inducibly activated by an external stimulus. It is also possible to use a vector having the same. The vector of the present invention preferably contains a gene expression regulatory mechanism and a terminator region. As the terminator region, a nopaline terminator, a 35S terminator, or the like can be incorporated.

  As one embodiment of the present invention, a binary vector can be used. A binary vector is a vector used in an Agrobacterium-mediated transformation method, and is a target foreign gene between T-DNA border sequences necessary for integration of T-DNA into chromosomal DNA of plant cells. Built to insert. On the other hand, a separate plasmid containing the vir region of the Ti plasmid necessary for the transfer of T-DNA into plant cells is constructed, and these two plasmids are introduced into Agrobacterium to infect the plant with this Agrobacterium. The desired foreign gene can be incorporated into the chromosomal DNA of the plant cell. As an example of such a binary vector, for example, pBI121 [EMBO. J. et al. 6, 3901-3907 (1987)]. pBI121 has a neomycin phosphotransferase gene and a GUS gene between the boundary sequences of T-DNA, and has a cloning site for insertion of a foreign gene between these genes, in E. coli and Agrobacterium. It is a binary vector that can be propagated. Examples of other binary vectors include pUCD2340 [E. Zyprian et al. , Plant Mol. Biol. 15, 245-256 (1990)]. pUCD2340 has an hph gene between the border sequences of T-DNA, and has a multiple cloning site downstream of the hph gene.

  Furthermore, in a preferred embodiment of the present invention, the recombinant vector further comprises a target gene to be expressed in the plant. As the target gene, various foreign genes can be introduced. In addition to the characteristics of the original plant, various characteristics due to the expression of the introduced foreign gene can be exhibited. For example, when the introduced foreign gene is a cold resistance gene, a transformed plant having cold resistance is provided. In the case of an agricultural chemical resistance gene, a transformed plant having resistance to a specific agricultural chemical is provided, and virus resistance is provided. In the case of genes, transformed plants can be provided that are resistant to infection with a particular virus.

<Plant transformation>
Transformation is a process in which genetic information held by a cell is altered by introducing one or more exogenous nucleic acids into the cell. For example, various methods known to those skilled in the art, such as polyethylene glycol method, electroporation method, Agrobacterium-mediated method, particle gun method, etc. (cell engineering separate volume, experimental protocol for new edition model plant, 2001, published by Shujunsha, etc.) and can be introduced into a plant or plant cell and transformed. For example, the in planta transformation method using the vacuum infiltration method first grows a healthy plant and induces a side branch by pinching after drawing. When the flower on the induced side branch begins to blossom, place the above-ground part of the plant body in a suspension of Agrobacterium with the recombinant DNA that you want to introduce and place it under a weak vacuum, and place it inside the flower of the suspension. Infiltration (reduced pressure infiltration). After infiltration under reduced pressure, Agrobacterium infection and T-DNA transfer occur, but only the T-DNA transfer that occurs in the flowers is transmitted to the next-generation seeds through fruiting, and is transformed at a certain frequency in the seeds. One generation (hemizygote) will be included. According to Clough, SJ and Bent, AF (1998), Plant J. 16: 735-743, decompression is not always necessary. In the floral dip method, as in the case of the vacuum infiltration method, the above-ground part of the plant body is immersed in the suspension of Agrobacterium, but the plant is lifted up by just immersing for about 3 to 5 seconds and lightly shaking. By increasing the concentration of the surfactant (Silwet L-77) in the infiltration suspension, a sufficiently high transformant frequency can be obtained without reducing the pressure.

  Tissue culture methods include polyethylene glycol method, electroporation method, Agrobacterium-mediated method, and particle gun method to introduce recombinant DNA into embryo-derived callus, and proliferate and redifferentiate this embryo-derived callus Let Any of these transformation methods can be used in the method of the present invention. However, the former in planta transformation method is preferred because it is simple and rapid and does not require the skill of an experimenter.

<Selection method using ethanol resistance as an index>
Mutant plants lacking the GEK1 gene are 10 to 100 times more sensitive to ethanol than wild type plants, but traits other than this ethanol sensitivity are no different from wild type. In other organisms, such as animals and microorganisms, such a mutant exhibiting such a large ethanol sensitivity is not known. In yeast, several ethanol-sensitive mutants have been obtained and their properties have been clarified, but these mutants are known to be involved in heat shock proteins and cell wall integrity. However, it has been found that the GEK1 gene according to the present invention is not related to any of these mechanisms.

  Therefore, according to the method of the present invention, as one form, an ethanol-sensitive plant or plant cell can be obtained by causing some mutation in the GEK1 gene, but it is not limited thereto. For example, the expression of the endogenous GEK1 gene may be suppressed as a result by factors other than the GEK1 gene such as expression of antisense RNA or introduction of siRNA. By introducing a recombinant DNA containing an exogenous GEK1 gene into these ethanol-sensitive plants or plant cells and culturing the transformed plant cells using a plant growth medium containing ethanol, the recombinant DNA is obtained. Can be selectively selected. In the present invention, the “plant cell” includes various forms of plant cells such as cultured cells, protoplasts, leaf sections, callus and the like. “Plant” refers to an individual plant composed of these plant cells or formed by proliferation and redifferentiation of these plant cells. Although various plant cells can be used as the transformed cells selected in the ethanol-containing medium by the method of the present invention, it is preferable to use the first generation seeds (hemizygotes) for transformation. This is because, unlike callus, etc., it can be stored temporarily in an easy state. Moreover, as specifically shown in the examples described later, transformants can be easily selected by collecting only seeds germinated on an ethanol-containing medium. The ethanol concentration in the medium can be efficiently selected in the range of 0.001 to 1% by volume, and the preferred concentration range is 0.01 to 0.1% by volume.

<Method for producing transformed plant>
In one embodiment of the present invention, a method for producing a transformed plant is provided. This method first comprises preparing a mutant plant in which the function of the plant endogenous GEK1 gene or its gene product is suppressed, introducing a recombinant DNA containing the GEK1 gene into the mutant plant, And the step of sowing seeds in a plant growth medium containing ethanol, and the transformed plant introduced with the recombinant DNA is selectively used in the ethanol-containing medium. Based on germination. Here, the function of the GEK1 gene or GEK1 gene product of the plant used as the host is preferably suppressed by 50% or more, preferably 70%, 80%, or 90% or more compared to the wild type, and most preferably Is the lack of the GEK1 gene. Such a mutant GEK1 gene can exist in various forms, such as a change in the primary structure of the GEK1 protein due to the addition, substitution or deletion of a base in the coding region. It is well known that substitution of one amino acid in a protein can greatly impair the function of the protein. Alternatively, it is also known that the amount and function of a gene product to be generated are greatly changed by causing a mutation in an expression regulatory region such as a promoter of GEK1 gene or an intron / exon boundary region.

  A method for producing such a mutant plant can be a method known to those skilled in the art and is not particularly limited. For example, mutagenesis treatment is performed by a chemical method, a physical method and a biological method. Select mutants. Mutagenesis is usually performed on seeds, and the mutagen-treated seeds are called M1 seeds and the grown plants are called M1 generation plants. The seed obtained by self-pollination from this plant is called M2 seed, and the plant grown from the seed is called M2 generation. In plants having a diploid genome, the gekl mutation generated in M1 seeds is observed for ethanol sensitivity only when M2 generation plants are examined.

  As chemical mutagenesis methods, alkylating agents such as EMS, nucleobase analogs, sodium azide and the like are used, and as physical methods, ionizing radiation, ultraviolet rays and the like are used. The biological method is a method of creating an insertion mutation by T-DNA or transposon. In plants in which the base sequence of the GEK1 homologous gene is unknown, surrounding DNA fragments can be isolated from the mutant obtained by this method using a transposon as a probe, and the GEK1 homologous gene can be cloned from the vicinity of the fragment ( Transposon tagging method). The mutant produced in this manner can easily isolate the gekl mutant by seeding seeds on a medium containing ethanol and confirming the presence or absence of germination.

  In another embodiment of the present invention, a wild type plant can be used instead of the mutant plant. In this case, the plant is transformed with an endogenous GEK1 gene present in the genomic DNA of the host plant and a GEK1 gene having a partially different base sequence, and the expression of the endogenous GEK1 gene is specifically determined. It should be suppressed. As such a suppression method, for example, siRNA or antisense RNA can be used.

  In still another embodiment of the present invention, a transformed plant obtained or obtainable by the transformation method is provided. A “transformant” or “transformed plant” is a cell or an aggregate or progeny thereof that has been genetically modified by incorporation of an exogenous nucleic acid, eg, a recombinant vector of the present invention. These terms also apply to progeny of transformed cells in which the genetic modification of interest is retained for several subsequent generations of cells, with or without other mutations or modifications.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1] Production of mutant strains of high-sensitivity ethanol The Arabidopsis thaliana Columbia type (Col: Columbia) or the Landsberg type (Ler: Landsberg erecta) is an MS medium (mixed salt for Murashige-Skoog medium) 2% (w / v) sucrose, 0.5 mM MES (pH 5.8), and 0.8% (w / v) agar) or in soil, 22-23 ° C., 16 hours / night Grow in an 8 hour cycle. Seeded seeds were kept at 4 ° C. for 3 or 4 days before incubating in the incubator to promote germination.

Fast neutron mutagenesis M ₂ seed, was purchased from LEHLE Seeds (Texas, USA). Ethyl methanesulfonate (EMS) treatment was performed as follows. Seed treated 0.3% (v / v) 16 hours at EMS at room temperature, after thorough washing with water, seeded in soil were harvested _{M 2} generation seeds. The _{M 2} seeds mutagenized, were plated on MS medium containing 0.04% (v / v) Various chemicals such as ethanol and abscisic acid. After incubation for 5-7 days, seeds that did not germinate were collected and transferred to MS medium for germination. From the mutant candidates obtained by such a method, namely, the EMS-treated Columbia-type M ₂ population, the fast-neutron mutagenized Columbia-type M ₂ population, and the EMS-treated Landsburg-type M ₂ population, Three independent ethanol hypersensitive mutants were identified: 1, gek1-2, and gek1-3. The isolated mutant was backcrossed 3 times.

As a result of analyzing the offspring obtained by test-crossing between the ethanol-sensitive strain (gek1) thus obtained and the wild-type strain, this mutation (gek1) is a recessive mutation. It was found to be encoded by a chromosomal gene (resistance: sensitivity = 307: 105, χ ² = 0.02, P = 0.89). As shown in FIG. 2 (A), none of the three gekl mutants could germinate in the presence of ethanol, but germinated normally in the absence of ethanol. The degree of ethanol sensitivity was different among the three alleles, with gek 1-3 being the most sensitive and gek 1-1 being the least sensitive. The germination rate of gek1-2 and gek1-3 mutants was significantly reduced in the presence of 0.003% (v / v, about 0.5 mM) ethanol, whereas the wild type was at least 0.3% (v / V) Germinated normally in the presence of ethanol (see FIG. 2 (B)).

[Example 2] Properties of ethanol-sensitive (gek1) mutant The phenotype of gekl is not limited to the germination stage. When seedlings having a gekl mutation are grown in normal medium and then transferred to a medium containing 0.03-0.1% (v / v) ethanol, their growth is markedly inhibited, and gek1- The 2 and gek1-3 mutants eventually died in the presence of 0.1% (v / v) ethanol. This gekl phenotype is specific for ethanol and its metabolites, methanol, butanol, isopropanol, dimethyl sulfoxide, paraquat, hydrogen peroxide, abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC, Ethylene precursor) or gibberellin did not affect germination and growth of the mutant. The high ethanol sensitivity of the gekl mutant was not affected by sunshine conditions or sucrose availability in the medium. No other phenotype could be found in the absence of ethanol.

  Since the gekl mutation is extremely ethanol-specific, the influence of ethanol on the metabolic pathway was examined. It is thought that ethanol in the medium is converted into toxic acetaldehyde by ADH in the cells. It has been reported that the ADH-deficient mutant of tobacco has enhanced ethanol tolerance, suggesting that acetaldehyde converted from ethanol by ADH is responsible for ethanol stress in plants. Therefore, it was confirmed whether there was a relationship between the gekl phenotype and the ADH activity.

  First, the position on the chromosome where the gekl mutation occurred was examined using a PCR-based genetic marker and found to be present at the top of the second chromosome. No gene involved in ethanol metabolism has been found at this position. Therefore, the genetic relationship between gekl and adh was examined. When the F2 generation obtained by performing test mating between these two recessive mutant strains was examined for ethanol sensitivity in germination, the separation ratio represented by the ratio of ethanol resistant strain and sensitive strain was 13: 3, It was shown that the adh mutation suppresses gekl. This result indicates that the ethanol-sensitive phenotype of gekl requires ADH activity.

[Example 3] Identification of gekl gene The Arabidopsis gekl gene was identified by chromosome walking based on a genetic map. Columbia-derived gek1-1 and gek1-2 were crossed with Ler to obtain a plurality of F ₂ progeny. Ethanol-sensitive strains were selected on a medium containing 0.1% (v / v) ethanol, and germinated and grown on a normal MS medium. Genomic DNA is extracted from these F ₂ progeny, and a marker based on PCR, for example, simple nucleotide sequence length polymorphism (Bell, C. and Ecker, JR (1994) Arabidpsis. Genomics 19: 137-144) is used. Mapping was performed. The results are shown in FIG. FIG. 3 (A) schematically shows the position and structure of the gekl gene on the chromosome. The numbers indicated by open triangles represent the recombination ratio that occurred between the genetic marker and the GEK1 gene. For example, the notation 5/1152 indicates that recombination occurred between this marker and the gekl gene in 5 out of 1152 second chromosomes examined. The smaller the number of recombination, the closer the marker is to the target gene. The mutation site of the gekl allele is indicated on the bottom bar. “Atg” indicates a translation initiation codon estimated by the Arabidopsis Genome Initiative (2000), and a small white triangle indicates the nucleotide sequence corresponding to the oligonucleotide primer used in the RT-PCR experiment shown in FIG. Indicates the position. FIG. 3 (B) shows GEK1 gene expression in various mutant strains. To amplify the 5 ′ half of the GEK1 coding region, two primers F19-25.1 (SEQ ID NO: 3) and F19-25.11 (SEQ ID NO: 4) were used, while to amplify the entire GEK1 coding region. RT-PCR was performed using two primers F19-25.1 (SEQ ID NO: 3) and F19-25.R (SEQ ID NO: 5), respectively. The two PCR products performed independently are shown in each lane of FIG. β-tubulin was used as a control.

F19-25.1: 5'-TTACGTTGGGAAGAAGCTACCG-3 '(SEQ ID NO: 3)
F19-25.11: 5'-CTGCCTCCTAGCCCAAGACC-3 '(SEQ ID NO: 4)
F19-25.R: 5'-ATGTTTTTAAGGGGGTCTCTCTAGA-3 '(SEQ ID NO: 5)
βTUB-F: 5'-ATCCCACCGGACGTTACAACG-3 '(SEQ ID NO: 6)
βTUB-R: 5'-TTCGTTGTCGAGGACCATGC-3 '(SEQ ID NO: 7)

As shown in FIG. 3, the GEK1 gene was localized on the BAC clone F19B11 of chromosome 2 (Arabidopsis Genome Initiative 2000). Independent gek1 alleles gek1-1, gek1-2, and gek1-3 were isolated from the three mutant strains prepared in Example 1. Furthermore, a different allele gek1-4 from another mutant strain was also isolated. The gek1-2 gene had a deletion from At2g03800 to At2g03810 at position 1977 counted from A of the putative translation start codon, and a stop codon was generated in frame adjacent to the deletion site. On the same gene, the single base substitution to A ^{G 135} in Gek1-1, single base substitution from ^{G 1685} in gek1-3 to A is, the single base substitution to A further gek1-4 the ^{G 1389} It was found. A single base substitution at gek1-1 causes an amino acid substitution from Met ⁴⁵ to Ile, a single base substitution at gek1-3 destroys the splicing acceptor site of the seventh intron, and a single base substitution at gek1-4 is Trp ²²⁹ Was changed to a stop codon (TGA), where the translation was terminated.

[Example 4] Isolation of GEK1 cDNA clone and genomic clone The GEK1 cDNA clone was isolated from a cDNA library constructed in the lambda Zap vector using a PCR amplified DNA fragment of the GEK1 genome as a probe. When the nucleotide sequence of the obtained cDNA clone was determined by the sequencer 3100 of Applied Biochemicals, it did not contain the estimated first ATG codon (Met ¹ ). The region up to the cleavage site was replaced with the corresponding region of genomic DNA, ie, the region from 83 bp upstream of the putative translation initiation codon to the MscI cleavage site. On the other hand, a 3.5 kb genomic clone containing the GEK1 gene is a high-precision Taq polymerase (KOD-Plus; Toyobo Co., Ltd.) using the following two types of oligonucleotides GEK1gEco (SEQ ID NO: 8) and GEK1gBam (SEQ ID NO: 9) as primers. Obtained by PCR. When the base sequence of the DNA fragment obtained by the PCR amplification reaction was determined, no change in the base sequence was observed.

GEK1gEco: 5'-GAGACGAATTCAATAGGTGGGTG-3 '(SEQ ID NO: 8)
GEK1gBam: 5'-ATATGGATCCGTGGACTTACTTGGTC-3 '(SEQ ID NO: 9)

[Example 5] Expression of GEK1 gene and conferring ethanol resistance An approximately 3500 bp chromosomal DNA fragment containing the GEK1 gene of Arabidopsis thaliana was inserted into the HIndIII-BamHI restriction enzyme recognition site of the Agrobacterium binary vector pBI101. Agrobacterium (Agrobacterium tumefacience, GV3101) was transformed with this recombinant plasmid (pNH677). For transformation, electroporation was used, and the bacterial solution after treatment was spread on an LB agar plate containing 25 mg / l kanamycin, and bacteria resistant to kanamycin were selected. The transformed Agrobacterium was cultured with shaking in LB liquid medium. The culture solution was centrifuged (5000 g, 10 minutes, 4 degrees) to precipitate the bacteria. The precipitated bacteria were suspended in Murashige sukuk medium (pH 5.8) containing 5% sucrose, 44 nM 6benzylaminopurine and 0.5 mM MES. 0.05% silwet L-77 was added to the bacterial suspension to obtain a bacterial solution for infection. Infection was carried out by immersing an Arabidopsis gek1-2 plant body having a plurality of flower buds in a bacterial solution for infection for several minutes. Infected Arabidopsis thaliana was grown to obtain first generation seeds. The obtained seeds were sown in a plant growth medium containing 0.1% ethanol to promote germination. As a result, germinating individuals were observed as shown in FIG. FIG. 4A is a photograph one week after inducing germination by sowing small brown seeds in the medium. FIG. 4B is an enlarged view thereof, and it can be seen that a green transformed plant (a young plant body in which leaves are expanded) grows, and germination of seeds that are not transformed is almost completely suppressed. Germinated individuals were grown, genomic DNA was extracted and the introduced genes were confirmed. As a result, a result suggesting that the GEK1 gene was inserted into the genome of the plant body germinated as expected was obtained.

It is the figure which compared and aligned the amino acid sequence of the GEK1-like protein of Arabidopsis thaliana, tomato, and rice. The symbols shown below the single letter abbreviations indicating amino acid residues are: * is an amino acid residue conserved in all three types of plants,: is a residue conserved with similar amino acid residues,. Indicates amino acid residues conserved in two of the three sequences, respectively. It is the graph (B) which shows the photograph (A) which germinated the seed of various mutants of Arabidopsis thaliana in the ethanol containing medium, and germination efficiency. ●: gek1-1, ■: gek1-2, ▲: gek1-3, ○: wild type, error bars indicate standard deviation of n = 4. It is the schematic diagram (A) which showed the mapping and structure of GEK1 gene, and the figure (B) which shows the result of having detected the expression of GEK1 gene in various mutant plants by RT-PCR. It is the result (A) and the enlarged view (B) which selected Arabidopsis thaliana into which GEK1 gene was introduced by germination in an ethanol containing medium.

Claims (11)

  A step of introducing a recombinant DNA containing the GEK1 gene into an ethanol-sensitive plant; a step of cultivating the plant to collect seed; and seeding the seed in a plant growth medium containing ethanol to germinate the transformed plant And a process for selecting a transformed plant.   The method according to claim 1, wherein the ethanol-sensitive plant has a mutation in a genomic endogenous GEK1 gene.   The method according to claim 1 or 2, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.   The method according to any one of claims 1 to 3, wherein the plant is sunflower, sunflower, potato, cotton, soybean, tomato, rice, leek, corn, barley, or wheat.   The GEK1 gene encodes a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a protein having at least 70% homology with the amino acid sequence and imparting ethanol tolerance to the plant The method according to any one of claims 1 to 4, comprising DNA.   The method as described in any one of Claims 1-5 in which the said culture medium for plant growth contains 0.001-1 volume% ethanol.   DNA comprising a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or DNA encoding a protein having at least 70% homology with the amino acid sequence and imparting ethanol tolerance to plants A plant transformation vector.   The transformation vector according to claim 7, further comprising a target gene to be expressed in the plant. Preparing a mutant plant in which the function of the plant endogenous GEK1 gene or its gene product is suppressed,
Introducing a recombinant DNA containing the GEK1 gene into the mutant plant;
Cultivating the plant and collecting seeds;
Sowing the seeds in a plant growth medium containing ethanol;
Selecting a plant germinated in the ethanol-containing medium, and a method for transforming the plant.   The method according to claim 9, wherein the recombinant DNA further comprises a target gene to be expressed in the plant.   A transformed plant, which has been transformed by the method according to claim 9 or 10.