JP2007104927A - Al101 gene of rice and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide the use of AL101 (Albino101) gene of rice by analyzing albino-mutant rice in the course of the investigation of DNA transposon nDart, confirming the destruction of the mutant caused by the insertion of nDart and identifying the mutable causal gene Al101 of mutation to form albino. <P>SOLUTION: Either one of the following genes is used for controlling the photosynthesis of vegetables: (1) a gene composed of a base sequence having a homology of ≥70% to a specific base sequence and (2) a gene composed of a specific amino acid sequence or an amino acid sequence obtained by the deletion, substitution or addition of one or more amino acids in the specific amino acid sequence and encoding a protein having activity to control the photosynthesis of vegetables. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

This patent application is mainly related to the results of commissioned research by the national government, etc. (2005 Agricultural and Biological Specified Industrial Technology Research Organization “Basic Research Promotion Project for Creating New Technology and New Fields”, Industry This is subject to Article 30 of the Revitalization Special Measures Law).
The present invention relates to rice AL101 gene, and more particularly to AL101 gene causing albino and a method for controlling photosynthetic ability by controlling expression of this gene.

Albino mutations without pigment are mutations that are frequently observed in nature, but are usually fatal. There are various albino causative genes, and they are caused by the deficiency of genes responsible for the functions essential for growth, but the functional analysis of the causative genes has not been sufficiently performed (for example, see Non-Patent Document 1). .
On the other hand, the present inventors have analyzed the cause of pyl (pale-yellow leaf) mutation, which reduces chlorophyll accumulation due to easy denaturation, and as a result, Ac / Ds-type transposon nDart, which has transfer activity under normal cultivation conditions in rice (Nonautonomous DNA-based active rice transposon) was confirmed (Patent Document 1).

Biochimie Volume 82, Issues 6-7, 7 June 2000, Pages 559-572 WO2005 / 003349

  In the course of conducting research on the DNA transposon nDart (Patent Document 1) discovered by the present inventors, the present inventors analyzed rice having albino mutations. That is, an object of the present invention is to identify a causative gene of an albino mutation, analyze its function, and provide a method for using it. A gene destroyed by a DNA transposon can be expected to recover the function of the destroyed gene due to its elimination. Therefore, the function identification of an unknown gene can be performed easily.

As a result of analyzing rice which is an easily degenerate albino mutant al101-m (albino101-mutable), the present inventors have found that in this mutant, transposon nDart (patent document 1) of the present inventors has already been found. After confirming that it was destroyed by insertion, the causative gene AL101 (Albino101) (SEQ ID NO: 1) of the mutation that easily converted to albino was identified.
This gene is located in the region of rice chromosome 4 at 24.8 Mbp (Pseudmolecules Build3, http://rgp.dna.affrc.go.jp/), and was named AL101 gene.
A gene destroyed by a DNA transposon can be expected to recover the function of the destroyed gene due to its elimination. Therefore, the function identification of an unknown gene can be performed easily. The present inventors identified a novel gene AL101 in rice and confirmed that this gene has a function of controlling plant photosynthesis.

That is, the present invention is the use of any of the following genes for controlling plant photosynthesis.
(1) a gene comprising a nucleotide sequence at least 70% homologous to the nucleotide sequence set forth in SEQ ID NO: 1 (2) the amino acid sequence set forth in SEQ ID NO: 2, or one or several amino acids in the amino acid sequence deleted, substituted or substituted A gene encoding a protein comprising an added amino acid sequence and having a plant photosynthesis control activity The present invention is a plant transformed by introducing any of these genes. Examples of the host include rice, Arabidopsis thaliana, tobacco, tomato, petunia, rape, cotton or corn.

  The albino gene AL101 of the present invention is considered to have an essential function for plant growth because its mutation is lethal in plants. By controlling the expression of this albino gene AL101, it is possible to improve the photosynthetic ability and produce a crop having a good growth state, thereby improving the yield and disease resistance.

  Albino is caused by a deficiency in genes responsible for growth, but many of them are lethal, so the functional analysis of the genes responsible for them has not been sufficiently performed. In the present invention, a causative gene (AL101) of a rice albino mutation was identified using an easily denatured strain capable of surviving the mutant. This AL101 gene is a novel gene having almost no homology with a known gene whose function has been analyzed.

The AL101 gene identified in the present invention is one of the following genes.
(1) a gene comprising a base sequence homologous to at least 70%, preferably 90%, the base sequence described in SEQ ID NO: 1 (2) the amino acid sequence described in SEQ ID NO: 2 or one or several amino acids in the amino acid sequence ( For example, a gene that encodes a protein having an amino acid sequence in which 5% of all amino acids are deleted, substituted, or added, and that has a plant photosynthesis control activity.

  As a criterion for automatically annotating genes in a genome sequence with a computer program (Otto system) in a report published by the Human Genome Consortium (Science 2001, 291 (5507), 1304-1351) When there is information, the reference for specifying the gene region in the genome from the homology with the sequence information is 92% or more in terms of the homology of the polynucleotide. In the same paper, 70% of the homology criteria for polynucleotides when searching for genes that have increased due to gene duplication (mutation has occurred in the subsequent evolution process and partial differences have occurred in the base sequence) are found. At. Therefore, in the present invention, if the homology with the nucleotide sequence of SEQ ID NO: 1 is 70% or more, preferably 90% or more, a gene having a plant photosynthesis control activity applied to a different but similar plant species. It is considered to function as.

The “plant photosynthesis control activity” can be confirmed as follows. A measurement construct in which the original promoter or an arbitrary promoter is connected upstream of the sequence whose control activity is desired is prepared. As a plasmid used for this construct, a binary vector such as Ti plasmid or pBI121 can be used. Using the prepared measurement construct, transformation is performed by a gene introduction method using Agrobacterium or a gene gun. The construction and transformation methods of this construct can be carried out using known “genetical recombination techniques” and “transformation methods”. Plants that have been successfully transformed can be selected, and the photosynthesis of the plants can be measured based on the growth status and the yield.
In a specific measurement, RNA is extracted from a part of the transformed plant, reacted with reverse transcriptase to synthesize cDNA, and quantitative RT-PCR is performed using this cDNA as a template to transform the transformed plant. Measure the expression level of the gene in When the gene expression level is low, the photosynthesis activity of the plant is low, and thus the control activity can be expressed by the gene expression level.

By controlling the expression of this AL101 gene, the photosynthetic ability of the plant can be improved.
For example, the expression level of the AL101 gene can be changed by replacing the promoter. Specifically, the gene is cloned into a general plasmid such as pBI121, the promoter is replaced, the gene is introduced into the plant by a transformation method using Agrobacterium, and a plant with an improved growth state is selected. be able to.

Moreover, RNA interference etc. are mentioned as a suppression method of gene function.
In addition, in an organism into which a part of the gene or cDNA obtained by reversely linking cDNA is introduced, the expression of the target gene can be suppressed (referred to as “suppression by antisense RNA”). In addition, when mRNA is expressed in large amounts by linking in the forward direction, it is recognized as a foreign substance and the degradation of mRNA is promoted, resulting in suppression of expression (“co-suppression technology” and “trans-witch technology”). "is called.). By using such a known technique for the gene of the present invention, the expression of the gene can be suppressed.

Moreover, a plant can be transformed using a plasmid introduced with these genes. As the plasmid, a binary vector such as Ti plasmid or pBI-121 plasmid can be used.
As the host plant, rice, Arabidopsis thaliana, tobacco, tomato, petunia, rape, cotton or corn are preferable. All have established transformation methods and are important plants in research, industry and horticulture.
When the AL101 gene is newly introduced into a wild-type plant, the ability to differentiate from pre-plastids to chloroplasts is increased, and the photosynthetic activity is increased, so that it can be expected to become a plant with high vitality. As a result, increased yield and improved disease resistance are expected.

The following examples illustrate the invention but are not intended to limit the invention.
Example 1
In this embodiment, albino mutant labile rice al101-m (albino101- m utable) a stable albino mutant al101-stb from progeny (al101- st a b le) was isolated (Figure 1) .
The new parting that this al101 mutant grew out of which included normal that did not show albino.
Ordinary albino mutants cannot grow beyond the third leaf stage, when the whole plant becomes albino and seed nutrition is lost. However, al101 mutants (al101-m, al101-stb) are inserted with DNA-type transposon nDart as shown in Examples 2 and 3 below, but nDart may be detached during growth. Yes, in this case, the al101 gene from which nDart has been released recovers its function and becomes normal. The new parting of the growth of the al101 mutant that does not show the above albino is an easily denatured phenotype with a green spot in the albino and is considered to be such a strain. Although it is an albino mutant that normally dies, it is thought that the green leaf portion compensates for the defect caused by the albino mutation by photosynthesis, so that the mutant can survive. This indicates that the AL101 gene has a function of controlling plant photosynthesis.

Example 2
The purpose of this example was to identify the causative gene for this al101 mutation.
nDart factors are known to have at least 60 copies distributed in the Nipponbare genome, and if all of them are amplified, the analysis becomes complicated, so the translocation activity is the highest and the cause of the al101 mutation NAiPCR, a modified version of the iPCR method (Trigrlia, T., Peterson, MG and Kemp, DJ (1988) Nucl. Acids Res. 16, 8186) to amplify only nDart1-O (NDart- homologues Non Amplified iPCR) method was established (FIG. 2).
The template used for NAiPCR was digested with 2.5 μg of genomic DNA of the al101-stb mutant with the restriction enzyme AluI, then treated with phenol / chloroform (P / C treatment), and the sample was self-ligated. Thereafter, the P / C-treated sample was digested with BmtI, and the P / C treatment was repeated to solubilize in 10 μl.
PCR reaction was performed in a 20 μl system using 2.5 μl as a template, and 1 x GC buffer, 250 μM dNTPS, 1 pM primer (5'-CGGGCCGTGCCGGCTACAGGTTC-3 '(SEQ ID NO: 4), 5'-CGTGCCGTGCTTGGGC-3' (SEQ ID NO: 4 5) The volume of LA-Taq 0.5 unit was adjusted with sterilized water, and the PCR reaction was 1 minute at 94 ° C, 1 minute at 65 ° C, 1 minute at 65 ° C, 3 minutes at 72 ° C. As a template for the second PCR reaction, 1 μl of a 100-fold diluted reaction product finally extended at 72 ° C. for 5 minutes after 20 cycles was used.
For the second PCR, use the primer set (5'-GCTACAGGTTCCGCCGTGCCTCGG-3 '(SEQ ID NO: 6), 5'-GGGCCGGCACGGCACGGCACAG-3' (SEQ ID NO: 7)) designed on the inside using the same system as the first PCR. It was. The PCR reaction is 94 ° C for 2 minutes, then 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 3 minutes, followed by 23 cycles, and finally extended at 72 ° C for 5 minutes to agarose gel (Fig. 3). In the NAiPCR method in lane 2 of FIG. 3, amplification of bands other than nDart1-O was suppressed by digestion with BmtI. As a result, amplification products of about 450 bp and about 270 bp were confirmed.

In order to confirm the PCR product amplified above, TA cloning was performed and the base sequence of the amplified product was determined. 1 μl of the above PCR product was cloned using a TA cloning kit (Invitrogen), the nucleotide sequence was determined using a sequencer (PRISM3100, Applied Biosystem), and the nucleotide sequence was analyzed using various software. As a result, 187 bp (62th to 248th positions of the sequence shown in FIG. 4B (SEQ ID NO: 12)) as the peripheral region of the site where nDart1-0 was newly inserted from the 270 bp amplification product (FIG. 4B, FIG. 3, SEQ ID NO: 12), As shown in Fig. 4A, 24.8Mbp (Pseudmolecules Build3, http://rgp.dna.affrc.go.jp/) region of rice chromosome 4 (full length 36.1Mbp), as shown in Fig. 4A Matched.
When a gene in this region was searched, the AK100471 clone (SEQ ID NO: 1) registered in the rice full-length cDNA database was detected, indicating that this gene is AL101. AL101 is an mRNA of approximately 2.1 Kbp, is composed of one exon, and has an open reading frame for a protein consisting of 628 amino acids (SEQ ID NO: 2).
As shown in FIG. 4B, nDart1-0 (SEQ ID NO: 3) is inserted into the promoter region 199 bp upstream from the translation initiation methionine of the AL101 gene. Usually, a region that functions as a promoter of a gene is considered to be 1 to 2 kb, and it was shown that the insertion of nDart1? O into this region is responsible for the albino mutation.

Example 3
In this example, the insertion of nDart confirmed in Example 2 was confirmed by transposon display, and it was reconfirmed that the cause of the albino mutation was the AL101 gene.
Al101-stb was used for analysis by the transposon display method. For the transposon display method, an adapter for restriction enzyme MseI (5′-GAGGATGAGTCCTGAG-3 ′ (SEQ ID NO: 8), 5′-CGCTCAGGACTCAT-3 ′ (SEQ ID NO: 9)) and primer (5′-GAGGATGAGTCCTGAGCGG-3 ′ ( SEQ ID NO: 10)) was used. Genomic DNA was digested with 1 μg of MseI, ligated with an adapter, and 8 ng was used as a template for PCR reaction. PCR reaction was carried out in a 20 μl system, and the volume was adjusted with sterile water to 1 × AdvantageGC Taq, 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 200 μM dTTP (BD), 0.48 μM primer set. The first PCR reaction is 94 ° C for 5 minutes, followed by 20 cycles of 94 ° C for 30 seconds, 48 ° C for 1 minute, 72 ° C for 2 minutes, and finally 72 ° C for 2 minutes. Then 0.2 μl was used as a template for the second PCR. In the second PCR, a primer (5′-AGTCCTGAGCGGCCCNTTTG-3 ′ (SEQ ID NO: 11)) labeled with rhodamine was used at 0.24 μM in order to selectively amplify the terminal sequence of the primer. PCR reaction is 94 ° C for 2 minutes, then 94 ° C for 30 seconds, 57 ° C for 30 seconds (reducing the temperature by 0.8 ° C per cycle), and 72 ° C for 1 minute per cycle for 13 cycles 30 cycles of 94 ° C. for 30 seconds, 48 ° C. for 30 seconds and 72 ° C. for 30 seconds were performed for 30 cycles, and finally the reaction was performed at 72 ° C. for 2 minutes. The reaction product was fractionated on a 5% acrylamide gel. The result is shown in FIG.
In FIG. 5, bands not present in wild type Taichung No. 65 were observed in al101-stb which is a stable albino mutant and al101-m which is an easily denatured albino mutant. When this band was cut out and the nucleotide sequence was determined, it was consistent with the al101 gene, and it was confirmed by the transposon display method that nDart was inserted into the al101 gene.

It is a photograph of an easily denatured albino mutant (al101-m) of rice. (1) shows the fruited al101 mutant (2) shows an enlarged photograph of the leaves of the al101 mutant. 1 is a diagram showing the principle of NAiPCR method performed in Example 1. FIG. Double lines represent transposons (nDart) and single lines represent peripheral sequences. The arrow indicates the primer and its amplification direction. As shown on the left of the figure, using the restriction enzyme AluI recognition region (AGCT) randomly distributed in the region surrounding nDart, self-ligated the digested genome and amplifies with primers designed inside nDart . In the case of NAi-PCR, as shown in the right side of the figure, digestion is performed using a recognition site for the restriction enzyme BmtI present in nDart1 other than nDart1-O to suppress amplification. It is a figure which shows the analysis result of al101-stb (stable albino mutant) by NAiPCR method. It is a figure which shows the structure of an albino gene (AL101), and the insertion site of nDart. A rectangular box represents an exon, and a black portion indicates a reading frame. The albino gene is located in the 24.8 Mbp region of rice chromosome 4, and a transposon (nDart, SEQ ID NO: 3) is inserted into the promoter region 199 bp upstream from the translation initiation methionine. B shows the amplification product (270 bp, SEQ ID NO: 12) of Example 2, and includes the peripheral region (62nd to 248th, 187 bp). Underlined parts (1-25th, 249-270th) indicate primers (SEQ ID NOs: 6, 7). A part (1-61st) of transposon nDart (SEQ ID NO: 3) is found upstream of the duplicated sequence (62-69th, black frame) generated by transposon insertion. Downstream of this sequence is the AL101 gene (SEQ ID NO: 1). It is a figure which shows insertion of the transposon (nDart) to the albino gene (AL101) by the transposon display method. 1 shows a band (425 bp) amplified only for al101-stb (stable albino mutant) and al101-m (easy-modifying albino mutant).

Claims (4)

Use of any of the following genes to control plant photosynthesis.
(1) a gene comprising a nucleotide sequence at least 70% homologous to the nucleotide sequence set forth in SEQ ID NO: 1 (2) the amino acid sequence set forth in SEQ ID NO: 2, or one or several amino acids in the amino acid sequence deleted, substituted or substituted A gene encoding a protein consisting of an added amino acid sequence and having a plant photosynthesis control activity Use according to claim 1, wherein the plant is rice, Arabidopsis, tobacco, tomato, petunia, rape, cotton or corn. A plant transformed by introducing any of the following genes:
(1) a gene comprising a nucleotide sequence at least 70% homologous to the nucleotide sequence set forth in SEQ ID NO: 1 (2) the amino acid sequence set forth in SEQ ID NO: 2, or one or several amino acids in the amino acid sequence deleted, substituted or substituted A gene encoding a protein consisting of an added amino acid sequence and having a plant photosynthesis control activity The plant according to claim 3, wherein the host is rice, Arabidopsis thaliana, tobacco, tomato, petunia, rape, cotton or corn.