JP2008253232A - High pigment accumulation plant - Google Patents

Abstract

An object of the present invention is to provide a highly pigment-accumulating plant.
A method for producing a pigment accumulation-transformed plant comprising the step of introducing a specific rice-derived gene into a plant.
[Selection figure] None

Description

  The present invention relates to a pigment high accumulation plant and a pigment synthesis regulatory gene, for example.

Plants produce a variety of natural products. One such natural product is pigment.
Non-Patent Document 1 discloses that a purple pigment is accumulated in a plant body due to overexpression of a specific gene. In the plant body, the expression of the phenylpropanoid biosynthesis gene is enhanced.
Thus, pigments accumulate in plants due to enhanced expression of genes involved in pigment synthesis. However, until now, no high accumulation plant of red pigment and / or ultraviolet absorbing pigment has been provided.

Justin O. Borevitz et al., "The Plant Cell", 2000, Vol. 12, p.2383-2393

  An object of this invention is to provide the pigment | dye high accumulation | storage plant which accumulate | stores a red pigment | dye, a ultraviolet-ray absorption pigment | dye, etc. in view of the situation mentioned above.

  As a result of diligent research to solve the above-mentioned problems, it was found that a highly pigment-accumulating plant can be produced by highly expressing a specific gene derived from rice (Oryza sativa) in the plant, and the present invention was completed. .

The present invention includes the following.
(1) A method for producing a pigment accumulation-transformed plant, comprising a step of introducing the gene described in (a) or (b) below into a plant.
(a) one or more genes selected from the group consisting of genes encoding proteins consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16;
(b) a gene encoding a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein (a), and imparting pigment accumulation ability to a plant (2) The method according to (1), wherein the dye is a red dye and / or an ultraviolet absorbing dye.
(3) A pigment accumulation-transformed plant produced by the method according to (1) or (2).

  According to the present invention, a method for producing a highly pigment-accumulating plant and a pigment accumulation-transformed plant by the production method are provided. Moreover, according to this invention, pigment high accumulation plants, such as a red pigment | dye and an ultraviolet-ray absorption pigment | dye, are provided.

Hereinafter, the present invention will be described in detail.
In order to select plants with high pigment accumulation, an Arabidopsis thaliana-rice FOX line in which rice full-length cDNAs are randomly overexpressed is prepared. Here, the Arabidopsis thaliana-rice FOX line is a general term for mutant strains in which rice full-length cDNAs are randomly overexpressed in Arabidopsis thaliana.

  Next, in order to select plants with high pigment accumulation from the plants of each line of the first (T1) generation of the Arabidopsis thaliana-FOX line, the true leaves were cut out from the T1 generation plants of the Arabidopsis thaliana-FOX line. The extract of the pigment extracted from the cut true leaves is applied to a microplate reader, and the absorbance at a predetermined wavelength absorbed by the pigment is measured. As a result of the measurement, a line that can be judged to have a significant accumulation of pigment as compared with a negative control (plant without gene transfer) is determined as a candidate for a highly pigment-accumulating plant. In this way, about 7,000 plants or more are selected, and then the pigment accumulation trait is inherited in the same manner as described above for the second generation (T2) generation plant candidate for pigment high accumulation plant. Check if it exists. As a result, the full-length cDNA of the introduced rice is determined using the PCR method and the DNA nucleotide sequence determination method for the strain that can be judged to have inherited pigment accumulation.

  In addition, retransformation of the full-length rice cDNA obtained into a plant was performed, and it was confirmed by the same method as above whether the trait with high pigment accumulation was reproduced in the obtained retransformant. I do.

  In this way, by confirming that the T1 generation and T2 generation of the transformant and the trait that accumulates the pigment in three stages with the retransformant are due to the full-length cDNA of rice introduced into the plant, It can be determined that the isolated full-length cDNA of rice confers high pigment accumulation ability on plants. In this way, a gene encoding a protein that imparts pigment accumulation ability can be isolated.

  As a result of the identification method described above, eight kinds of rice-derived genes conferring high pigment accumulation ability were isolated. The genes are shown in Table 1 below.

  Table 1 shows the strain number in the rice FOX database from which each gene is derived, the registration number in the Institute for Genomic Research (TIGR) in the United States (in the column of “rice cDNA” in Table 1), and the annotation on the protein encoded by each gene ( Expected function), as well as the cDNA of each gene and the corresponding amino acid sequence SEQ ID NO.

  Based on the above findings, it was found that pigments can be highly accumulated in plants by introducing and expressing these genes in plants.

  That is, the method according to the present invention comprises a step of introducing a pigment accumulation-transformed plant comprising a step of introducing one or more (1, 2, 3, 4, 5, 6, 7 or 8) genes listed in Table 1 into a plant. It is a manufacturing method.

  In the present invention, genes to be introduced into plants (hereinafter referred to as “transgenes”) are those listed in Table 1. That is, a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 is a transgene in the present invention. In addition, in the amino acid sequence of the protein corresponding to each gene shown in Table 1, one or several (for example, 1 to 10, preferably 1 to 5, particularly preferably 1 to 3) amino acids are deleted or replaced. Alternatively, a gene consisting of an added amino acid sequence and encoding a protein that imparts pigment accumulation ability to a plant can also be used as a transgene in the present invention. Furthermore, the transgene in the present invention includes the amino acid sequence of the protein corresponding to each gene listed in Table 1, and 80% or more, preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more. Furthermore, a gene encoding a protein consisting of an amino acid sequence that is preferably 99% or more identical and imparting pigment accumulation ability to a plant is also included.

  Or the gene of Table 1 is DNA which consists of a base sequence shown by sequence number 1, 3, 5, 7, 9, 11, 13 or 15. Therefore, in the base sequence of each gene described in Table 1, one or several (for example, 1 to 10, preferably 1 to 5, particularly preferably 1 to 3) bases are substituted, deleted or added. DNA that encodes a protein comprising a base sequence and that imparts a pigment accumulation ability to plants, and a DNA comprising a base sequence complementary to the DNA comprising the base sequence of each gene listed in Table 1 under stringent conditions 80% or more, preferably 90% or more, 95% or more, 96% or more, 97% or more of the DNA encoding the protein that is soybean and imparts pigment accumulation ability to the plant and the base sequence of each gene shown in Table 1 As described above, the transgene according to the present invention also includes a DNA comprising a base sequence that is 98% or more, particularly preferably 99% or more, and that encodes a protein that imparts pigment accumulation ability to a plant.

  In addition, as long as the gene has the above sequence characteristics, not only rice but also genes derived from other plant species are included in the transgene in the present invention.

  Here, `` pigment accumulation ability '' refers to the entire plant body, plant organs (e.g. leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g. epidermis, phloem, soft tissue, xylem, vascular bundles, etc.) It means the ability to accumulate pigments in plant cultured cells. Measurement of the pigment accumulation ability of the protein encoded by the transgene to the plant is performed, for example, by using the extract of the pigment extracted from the plant (transformant) into which the above-described transgene has been introduced to an absorptiometer or a microplate reader, This is done by measuring the absorbance at a predetermined wavelength that the dye absorbs. The above-mentioned pigment extract is obtained by, for example, immersing a plant in a pigment extract (80% methanol, 10% hydrochloric acid) for 2 days to remove the plant. The extraction step is preferably performed at 4 ° C. with light shielding. In this way, when the accumulation of pigment is significantly observed in the transformant compared to the negative control (plants into which the above-described transgene has not been introduced), the above-mentioned transgene has the ability to accumulate pigment. It can be determined.

  The stringent conditions described above are, for example, when using probe DNA labeled with phosphorus 32, 5 × SSC (0.75M NaCl, 0.75M sodium citrate), 5 × Denhardt's reagent (0.1% Ficoll, 0.1% In a hybridization solution consisting of 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin) and 0.1% sodium dodecyl sulfate (SDS), the temperature is 45 ° C. to 65 ° C., preferably 55 ° C. to 65 ° C. In the washing step, the temperature is 45 ° C. to 55 ° C. in the washing solution consisting of 2 × SSC and 0.1% SDS, more preferably 45 ° C. to 55 ° C. in the washing solution consisting of 0.1 × SSC and 0.1% SDS. It is. When using an enzyme-labeled probe DNA using a kit of AlkPhos direct labeling module (Amersham Biotech), a hybridization solution having a composition described in the kit manual (including 0.5 M NaCl and 4% blocking reagent) The temperature is 55 to 75 ° C. In the washing step, the temperature is 55 ° C. to 75 ° C. in the primary washing solution (containing 2M urea) described in the manual of the kit and the room temperature in the secondary washing solution. In addition, other detection methods may be used, and in that case, standard conditions of the detection method may be used.

  On the other hand, in the present invention, examples of pigments accumulated in plants include red pigments (wavelength range 490 nm to 550 nm, preferably 510 nm to 530 nm) and ultraviolet absorbing pigments (wavelength range 290 nm to 350 nm, preferably 290 nm to 320 nm). It is done.

  In the method according to the present invention, first, a transgene is prepared. For example, a transgene can be obtained by PCR using rice genomic DNA or cDNA as a template and appropriate primers designed for a given gene. Moreover, the form of the transgene for introduction into a plant may be, for example, DNA containing the transgene or expression vector containing the transgene. The vector may be any vector that can be used for plant transformation, and examples thereof include Agrobacterium Ti plasmid or Ri plasmid. An expression vector containing a transgene can be obtained by inserting the transgene into a multicloning site of an appropriate vector. In addition, it is preferable that the expression vector appropriately includes a control region such as a promoter, enhancer, and terminator, a selection marker such as a drug resistance gene, and the like. When the dye is to be accumulated in a specific cell, tissue, organ or the like, a promoter specific for the cell, tissue or organ is introduced into the vector at a position where the transgene is controlled. As described above, when the expression vector contains a specific promoter and transgene, in the introduced plant (transformant), the transgene is specifically expressed in a predetermined cell, tissue, or organ, so that As a result, the dye can accumulate in the cell, tissue or organ.

Next, in the method according to the present invention, a transgene or an expression vector containing the transgene is introduced into a plant, and the plant is transformed. In the present invention, “plants” to be introduced include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, etc.). Or, it means any plant cultured cell. The host plant may be any plant, but examples thereof include monocotyledonous plants and dicotyledonous plants such as plants belonging to the family Gramineae, Brassicaceae, Solanum, Legumes (see below).
Gramineae: rice, maize (Zea mays)
Brassicaceae: Arabidopsis family: Tobacco (Nicotiana tabacum)
Legumes: Soybean (Glycine max)

  A transgene or an expression vector containing the transgene can be introduced into a plant by a general transformation method such as electroporation, Agrobacterium, particle gun, or PEG.

  For example, when the electroporation method is used, an electroporation apparatus equipped with a pulse controller is used to treat a voltage of 500 to 1600 V, 25 to 1000 μF, and 20 to 30 msec. It can be introduced into a host plant.

  When the particle gun method is used, the whole plant, plant organ, and plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The prepared sample can then be processed using a gene transfer device (for example, PDS-1000 / He from Bio-Rad). Treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 1000 to 1800 psi and a distance of about 5 to 6 cm.

  In the Agrobacterium method using Agrobacterium Ti plasmid or Ri plasmid, when a bacterium belonging to the genus Agrobacterium infects a plant, it has the property of transferring a part of the plasmid DNA that it has into the plant genome. Utilizing it, the transgene is introduced into the host plant. Among bacteria belonging to the genus Agrobacterium, Agrobacterium tumefaciens infects plants to form a tumor called crown gall. On the other hand, Agrobacterium rhizogenes infects plants and generates hairy roots. These are due to the fact that a region called T-DNA region (Transferred DNA) on the Ti plasmid or Ri plasmid is transferred into the plant and integrated into the plant genome during infection. Therefore, the transgene to be integrated into the plant genome is inserted into the T-DNA region on the Ti or Ri plasmid. Subsequently, when the host plant is infected with a bacterium belonging to the genus Agrobacterium, the transgene can be incorporated into the plant genome.

  Tumor tissue, shoots, hairy roots and the like obtained as a result of transformation can be used as they are for cell culture, tissue culture or organ culture. Further, it can be regenerated into a plant body (transformant) by administration of a plant hormone or the like using a conventionally known plant tissue culture method.

  In addition, a transgene can be introduced into a plant by using a plant virus as a vector. Examples of plant viruses that can be used include cauliflower mosaic virus. First, the virus genome is inserted into a vector derived from E. coli to prepare a recombinant, and then the transgene is inserted into the virus genome. The transgene can be introduced into the host plant by excising the viral genome thus modified from the recombinant with a restriction enzyme and inoculating the host plant.

  Further, it can be confirmed whether or not the transgene has been incorporated into the host plant by PCR, Southern hybridization, Northern hybridization or the like. For example, DNA is prepared from the transformant, and PCR is performed by designing primers specific to the transgene. Next, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, whereby Confirm that it has been converted. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, a method may be employed in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction.

  By growing the transformant thus obtained, a highly pigment-accumulating plant (pigment-accumulating transformed plant) can be obtained. The highly pigment-accumulating plant obtained by the method according to the present invention is a visually altered (red line) plant. In addition, a highly pigment-accumulating plant obtained by the method according to the present invention can grow in a region with higher illuminance than a non-transformed plant by accumulating ultraviolet absorbing pigment. Furthermore, the traits of highly pigment-accumulating plants obtained by the method according to the present invention are significantly passed on to the next generation.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Example 1] Search for a gene encoding a protein that imparts pigment accumulation ability First, to select plants that highly accumulate pigment, Arabidopsis thaliana-rice FOX line in which rice full-length cDNA was randomly overexpressed Was made. The production method of the Arabidopsis thaliana-rice FOX line was as follows.

  Full-length cDNA was prepared from rice by the CAPtrapper method. Next, the obtained cDNA was cloned into a site sandwiched between SfiI restriction enzyme sites of Lambda PS. Furthermore, cDNA grouping was performed by reading the base sequence of the cloned cDNA using the vector sequence, and 13,000 independent clones were identified.

  Next, an equal amount of each clone adjusted to an equal concentration was dispensed and mixed into one tube. This is called “normalized full-length rice cDNA mix”.

  The normalized full-length cDNA mix and Ti plasmid pBIGS2113-SF were mixed and then completely cleaved with SfiI. After cleavage, a ligation reaction with T4 ligase was performed, and the reaction solution was transformed into E. coli. Next, about 130,000 independent colonies grown on agar medium containing kanamycin (Km) were mixed, and the plasmid was recovered therefrom.

  The recovered plasmid was then transformed into Agrobacterium GV3101. After transformation, 150,000 independent colonies grown on Km-containing agar medium were suspended in LB liquid medium, glycerol was added to a concentration of 15% in the medium, and the mixture was stored at -80 ° C. This glycerol solution is called “FOX Agrobacterium Library”.

  This FOX Agrobacterium library was grown to about 200,000 colonies, suspended in a transformation medium, and then transformed into wild-type Arabidopsis thaliana (Ecotype: Colombia). Then, the seeds were harvested, and the obtained seeds were sown on a hygromycin-containing BAM medium, grown under normal conditions, and only hygromycin-resistant plants were transplanted to the soil. The transformed Arabidopsis thus grown was designated as the T1 generation of the Arabidopsis-rice FOX line.

  On the other hand, in order to select plants with high pigment accumulation from plants of each T1 generation of the Arabidopsis thaliana-FOX line, we developed a method for selecting highly efficient pigment high accumulation plants. The reason is that the selection of the T1 generation of the Arabidopsis thaliana-FOX line is the most efficient, so the pigment accumulation amount of the plant is measured with high efficiency equal to or higher than the production speed of the T1 generation of the Arabidopsis thaliana-FOX line. This is because the way to do it was indispensable. As a result of various methods, it was found that the selection method using a microplate reader is the most efficient. The selection method involves cutting the true leaf from the T1 generation plant of the Arabidopsis thaliana-FOX line, using the extract of the pigment extracted from the cut true leaf in a microplate reader, and measuring the absorbance at a predetermined wavelength absorbed by the pigment. It is a method of measuring.

  Therefore, the true leaves were cut from plants of each of more than about 7,000 strains of the T1 generation of the Arabidopsis-rice FOX line described above.

  Subsequently, the pigment | dye was extracted from the cut true leaf. In the pigment extraction, a plant extract was immersed in a pigment extract (80% methanol, 10% hydrochloric acid) or the like for 2 days under light shielding at 4 ° C. to remove the plant body, thereby obtaining a pigment extract.

  The obtained dye extract was subjected to a microplate reader, and the absorbance was measured at wavelengths of 305 nm (ultraviolet absorbing dye) and 530 nm (red dye).

  As a result of the absorbance measurement, several plants were isolated that could be judged to have significantly accumulated UV-absorbing pigment and red pigment as compared to the negative control (plant without gene transfer). Some of these plants were visually observable with high pigment accumulation (Fig. 1). Further, for these T2 generation plants, it was confirmed whether or not the trait in which the ultraviolet absorbing dye and the red dye were accumulated was inherited by the same method as described above. As a result, the full-length cDNA of the introduced rice was determined using the PCR method and the DNA nucleotide sequence determination method for the lines that could be judged to have inherited the accumulation of the pigment.

  In this way, eight types of genes encoding proteins that impart pigment accumulation ability were obtained. The obtained genes are shown in Table 2 below.

  Table 2 shows the strain number in the rice FOX database from which each gene is derived, the registration number in the Institute for Genomic Research (TIGR) in the United States (in the column of “rice cDNA” in Table 2), and the annotation on the protein encoded by each gene ( Expected function), as well as the cDNA of each gene and the corresponding amino acid sequence SEQ ID NO.

  On the other hand, FIG. 1 shows a representative example of a visual phenotype of pigment accumulation. In FIG. 1, a photograph of A is a phenotype of a transformant into which a gene (cDNA) having the base sequence shown in SEQ ID NO: 13 (line number K03036) has been introduced. The photograph of B shows the phenotype of the transformant into which the gene (cDNA) (line number K00851) consisting of the base sequence shown in SEQ ID NO: 1 has been introduced. The photograph C shows the phenotype of the transformant into which the gene (cDNA) consisting of the base sequence shown in SEQ ID NO: 9 (line number K06951) was introduced.

  As shown in FIG. 1, it can be seen that the appearance of the pigment greatly changes due to the high accumulation of the dye, depending on the accumulation amount.

[Example 2] Production of a retransformant having a gene encoding a protein that imparts pigment accumulation ability A gene (cDNA) encoding a protein that imparts pigment accumulation ability obtained in Example 1 was transformed into a plant ( It was confirmed by the same method as in Example 1 whether or not the trait in which the pigment was highly accumulated was reproduced in the retransformant obtained after retransformation into Arabidopsis thaliana. Confirmed that the transformants of Example 1 T1 and T2 generations and the retransformants of this Example and the trait in which pigments accumulate in three stages are close to the full-length cDNA of rice introduced into the plant Thus, it was determined that the rice full-length cDNA isolated in Example 1 confers high pigment accumulation ability to plants.

  Each gene (cDNA) shown in Table 2 was amplified by the PCR method using rice cDNA as a template and a specific primer having a restriction enzyme SfiI site added to the end.

  Next, each amplified gene was incorporated into the SfiI site of Ti plasmid pBIGS2113-SF for plant expression. Agrobacterium tumefaciens was transformed with the plasmid incorporating each gene.

  The Agrobacterium obtained by the transformation was cultured with shaking at 28 ° C. overnight in LB medium (1% tryptone / peptone, 0.5% yeast extract, 1% NaCl).

  After shaking culture, the cells were resuspended in a transformation medium (2.7 g / L MS Salt, 112 μl / L V5 Vitamines, 50 g / L Sucrose, 200 μl / L Silicone L-77). The stem of the Arabidopsis thaliana after the lottery was immersed in the obtained suspension, and the plant body was put in a plastic bag and allowed to stand overnight. After standing overnight, the plant was removed from the plastic bag and allowed to grow under normal conditions until seed collection.

Then, the obtained seeds were sown in an antibiotic-containing BAM medium (0.1 g / L KNO ₃ , 8 g / L bacto agarose, 20 mg / L hygromycin B) and grown under normal conditions. Plants resistant to antibiotics were observed to attach true leaves on the medium in about two weeks. In addition, the antibiotic-resistant plants were transplanted into soil and grown under normal conditions. This plant is not only resistant to antibiotics but also a transformant in which each gene (cDNA) shown in Table 2 is integrated into the Arabidopsis genome.

  In each transformant, when the protein encoded by the gene is excessively synthesized in the plant due to the influence of the promoter upstream of each gene shown in Table 2, the absorbance is measured using a microplate reader. Thus, it was confirmed whether or not the pigment was significantly accumulated as compared with the negative control (plant without gene transfer) (FIGS. 2 and 3).

  FIGS. 2 and 3 show the measurement results of the ultraviolet absorbing dye and red dye accumulation in the second generation (T2) of the retransformants obtained by transformation using each transgene.

  2 and 3, each A graph shows the measurement results of absorbance at a wavelength of 305 nm (ultraviolet absorbing dye). On the other hand, each B graph shows the measurement results of absorbance at a wavelength of 530 nm (red pigment). 2 and 3, retransformants into which each transgene has been introduced are indicated by line numbers. The negative control (Col.) is the result of a plant that has not been transfected. Further, each bar graph within the same line number is the result of a separate T2 generation plant obtained from the same T1 generation retransformant.

  As shown in FIGS. 2 and 3, for all of the retransformant lines shown here, more than half of the plants were significantly accumulated with UV-absorbing dye and red dye compared to the negative control. . This result indicates that the phenotypic trait in which the pigment is highly accumulated is dominant, and strongly suggests that the protein encoded by each introduced gene is due to excessive synthesis.

FIG. 1 shows a representative example of a visual phenotype of pigment accumulation. FIG. 2 shows the results of measurement of ultraviolet absorbing dye and red dye accumulation in the second generation (T2) of retransformants obtained by transformation using each transgene. FIG. 3 shows the measurement results of the accumulation of UV-absorbing dye and red dye in the second generation (T2) of the retransformant obtained by transformation using the transgene (rice cDNA (Os07g20270; SEQ ID NO: 15)). .

Claims (3)

A method for producing a pigment accumulation-transformed plant, comprising the step of introducing the gene described in (a) or (b) below into a plant.
(a) one or more genes selected from the group consisting of genes encoding proteins consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16
(b) a gene encoding a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the protein of (a), and imparting pigment accumulation ability to a plant   The method according to claim 1, wherein the dye is a red dye and / or an ultraviolet absorbing dye.   A pigment-accumulating transformed plant produced by the method according to claim 1 or 2.

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