CN103740732B - Rice yellowing-to-greeninmarker marker gene and application thereof - Google Patents

Abstract

The invention discloses a rice yellowing-turning-greening marker gene and application thereof. The invention provides rice yellow-to-green mutant gry79, and its preservation number is CGMCC NO.8769. Also provided is the mutant gene mGRY79 in the rice yellow-to-green mutant gry79. Application of the above-mentioned rice yellowing-turning-green mutant gry79 or the above-mentioned mutant gene mGRY79 in cultivating rice male sterile lines with yellowing-turning-green traits; the experiment of the present invention proves that using chemical mutagenesis to obtain rice yellowing-turning green mutants gry79. Comparing wild-type and mutants, a new gene GRY79 was found in wild-type rice, and its corresponding mutant gene was found in mutants. The mutant gene can be used as a leaf color marker gene for early effective identification and efficient removal of hybrid rice seedlings Pseudo-hybrids, thereby improving the field purity of hybrid rice.

Description

Marker gene for yellowing and greening in rice and its application

technical field

The invention relates to the field of biotechnology, in particular to a rice yellowing-turning-greening marker gene and application thereof.

Background technique

Plant leaf color is a comprehensive expression of various pigments in chloroplasts. In normal leaves, chlorophyll is dominant, usually appearing as green. Leaf color mutation is a relatively common mutation type in rice, which is generally controlled by recessive nuclear genes. Dozens of rice leaf color mutants have been reported, which can be roughly divided into several categories such as yellowing, albinism, stripes, and spots. The distribution of leaf color mutation genes has been found on each chromosome of rice. The molecular mechanism of leaf color mutation is relatively complex. Mutant genes can cause leaf color variation through a variety of pathways, such as chlorophyll biosynthesis-related gene mutation, chloroplast differentiation and development blockage, cytoplasmic-nuclear signal transduction pathway blockage, and feedback regulation disorder of heme. , the regulation of phytochrome is blocked, the transport of chloroplast protein is blocked, etc. In short, the mutant gene directly or indirectly affects the synthesis and degradation of photosynthetic pigments, especially chlorophyll, resulting in changes in the content and proportion of various pigments, thereby causing leaf color variation. For example, rice chlorophyll synthase gene YGL1 and ethylene reductase gene OsDVR are involved in chlorophyll biosynthesis, and mutations in these two genes cause leaf yellowing (Wu ZM, Zhang X, He B, Diao LP, Sheng SL, Wang JL, Guo XP, Su N, Wang LF, Jiang L, Wang CM, Zhai HQ, Wan JM(2007) A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiol145:29-40; Wang PR, Gao CM JX, Wan , Zhang FT, Xu ZJ, Huang XQ, Sun XQ, Deng XJ(2010)Divinyl chlorophyll(ide)a can be converted to monovinyl chlorophyll(ide)a by a divinylreductase in rice.Plant Physiol153:994-1003); The stripe gene V2 gene encodes guanylate kinase, which is involved in catalyzing the conversion of GMP to GDP in the guanylate metabolic pathway in plastids and mitochondria, and the mutant gene has a delaying effect on the early development of chloroplasts (Sugimoto H, Kusumi K, Noguchi K, Yano M, Yoshimura A, Iba K (2007) The rice nuclear gene, VIRESCENT2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria. Plant J52:512-527).

The two-line hybrid rice based on photothermosensitive male sterility has been widely used in production, and has made great contributions to the development of rice production and hybrid rice technology in my country. However, the fertility expression of photothermosensitive GMS lines is affected by environmental conditions, especially temperature. In some years, due to unpredictable abnormal weather conditions, fertility fluctuations lead to hybrid seeds produced in seed production fields containing more The self-bred seeds of the sterile line (ie "pseudo-hybrid") will bring significant losses to seed production and field production. Leaf color marking can be used to identify the seed purity of hybrid rice, especially two-line hybrid rice, to efficiently eliminate false hybrids, improve the purity of hybrids, and prevent possible major losses to production, so it has attracted attention. The leaf color marker traits used in hybrid rice breeding should meet the following four conditions: (1) the marker traits are obvious, the expression period is early, and the seedlings are easy to identify; (2) the marker traits are stable and not easily affected by environmental factors; (3) the marker traits The traits are controlled by recessive nuclear genes; (4) The marker traits have no significant negative effect on the yield of hybrids or sterile lines. Among them, the mutation trait of yellowing (or albinism) turning green is particularly advantageous in solving the problem of hybrid rice seed purity as a leaf color marker trait. On the one hand, it can realize early, convenient and accurate identification of hybrid purity; Breeding line, the seedlings grow slowly in the early stage, and the group competitiveness is weak. If they are mixed in hybrids, they are easy to be shaded and die, so as to efficiently eliminate false hybrids (maternal sterile lines) and improve the purity of hybrids; Due to the weak photosynthesis, the growth period expression may affect the agronomic traits of the sterile line itself and its reproduction and seed production yield, so it is more valued and utilized by breeders (Wu D X, Shu Q Y, Xia Y W.In vitro mutagenesis induced novelthermo/photoperiod sensitive genic male sterile indica rice with green-revertible xanthanleaf color marker.Euphytica,2002,123:195-202; Breeding and characteristics of the genetically sensitive male sterile line Yutu S. China Rice Science, 18(6):515-521; Leaf color marker two-line hybrid rice male sterile line NHR111S. Journal of Nuclear Agriculture, 20(2):103-105).

Recently, it has been reported in the literature that the rice seedling stage albinism-to-green mutant gene ysa can be used as a marker gene for early effective identification of two-line hybrid rice seedlings and efficient removal of pseudo-hybrids, thereby improving the field purity of two-line hybrid rice (Su N, HuML, Wu DX,Wu FQ,Fei GL,Lan Y,Chen XL,Shu XL,Zhang X,Guo XP,Cheng ZJ,LeiCL,Qi CK,Jiang L,Wang HY,Wan JM(2012)Disruption of a rice pentatricopeptide repeatprotein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production. Plant Physiol 159:227-238).

Contents of the invention

An object of the present invention is to provide a DNA molecule.

The DNA molecule provided by the present invention (that is, the mutant gene mGRY79) is any one of the following 1)-4):

1) The coding region is the DNA molecule shown at position 10-7501 from the 5' end of sequence A, the sequence A is a sequence obtained by mutating sequence 1 from the 4218th base C at the 5' end to T in the sequence listing;

2) The coding region is the 10th-2625th position from the 5' end of sequence B, the sequence B is the mutation of sequence 2 in the sequence listing from the 1169th base C to T at the 5' end;

3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes a protein with the same function;

4) A DNA molecule that has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in 1) or 2) and encodes a protein with the same function.

The above stringent conditions are hybridization at 65°C in a solution of 6×SSC, 0.5% SDS, and then washing the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

Another object of the present invention is to provide rice yellow-to-green mutant gry79.

The rice yellow-to-green mutant gry79 provided by the present invention has a preservation number of CGMCC NO.8769.

The application of the above-mentioned DNA molecule (that is, the mutant gene mGRY79) or the above-mentioned rice yellowing-turning-green mutant gry79 in breeding rice male sterile lines with yellowing-turning-green traits is also within the protection scope of the present invention.

The conventional hybrid rice parent sterile line can be crossed and/or backcrossed with the rice yellowing-turning-green mutant gry79 according to a conventional breeding method, so as to obtain a rice male sterile line with the yellowing-turning-green trait.

In the above-mentioned application, the trait of yellowing and turning green is that the leaves of the plant are yellow-green in the 1-leaf stage-3 leaf stage, and the leaves gradually turn green from the 4-leaf stage, and the leaves are green in the 6th leaf stage-heading and fruiting stage; the plant is rice;

In the above application, the rice is specifically japonica rice or indica rice.

The invention also provides a method for cultivating male sterile lines of rice with the trait of yellowing and turning green.

The method provided by the present invention is to use the above-mentioned rice yellowing-turning-green mutant gry79 as a donor and the hybrid rice parent male sterile line as a recipient to perform hybridization and/or backcrossing to obtain a yellowish-turning-green mutant. Rice male sterile line;

The yellowing-to-green trait is that the leaves of the plant are yellow-green in the 1-leaf stage-3 leaf stage, and the leaves gradually turn green from the 4-leaf stage, and the leaves are green in the 6th leaf stage-heading and fruiting stage; the plants are rice;

The rice is specifically japonica rice or indica rice.

The application of the above-mentioned DNA molecule (that is, the mutant gene mGRY79) in assisting in identifying whether the plant to be tested is a yellow-turned-green plant.

The above application is to detect the genomic DNA or cDNA of the plant to be tested, if the genome of the plant to be tested contains the DNA molecule (genomic DNA of the mutant gene mGRY79) shown in 1) of the above DNA molecules, or the cDNA of the plant to be tested contains the above DNA If the DNA molecule (cDNA of the mutant gene mGRY79) shown in 2) in the molecule (cDNA of the mutant gene mGRY79), the plant to be tested is or is a candidate for a yellowish-turned-green plant, if the genome of the plant to be tested does not contain the DNA shown in 1) in the above DNA molecule If the molecule or the cDNA of the plant to be tested does not contain the DNA molecule shown in 2) in the DNA molecule above, the plant to be tested is not or the candidate is not a yellow-turned-green plant;

The yellowing-to-green trait is that the leaves of the plant are yellow-green in the 1-leaf stage-3 leaf stage, and the leaves gradually turn green from the 4-leaf stage, and the leaves are green in the 6th leaf stage-heading and fruiting stage; the plants are rice;

The rice is specifically japonica rice or indica rice.

Another object of the present invention is to provide a protein.

The protein provided by the present invention is as follows (a) or (b):

(a) A protein consisting of the amino acid sequence shown in Sequence 3 in the Sequence Listing;

(b) A protein derived from Sequence 3 in which the amino acid sequence shown in Sequence 3 in the Sequence Listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and is related to plant chlorophyll content.

The above-mentioned substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The DNA molecule (GRY79 gene) encoding the above protein is also within the protection scope of the present invention.

The DNA molecule (GRY79 gene) is specifically any one of the following 1)-4) DNA molecules:

1) The coding region is the DNA molecule indicated by the DNA molecule indicated by the 10th-2625th nucleotide from the 5' end of Sequence 2 in the sequence listing;

2) The coding region is the DNA molecule shown in nucleotides 10-7501 from the 5' end of sequence 1 in the sequence listing;

3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes a protein with the same function;

4) A DNA molecule that has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in 1) or 2) and encodes a protein with the same function.

The above stringent conditions are hybridization at 65°C in a solution of 6×SSC, 0.5% SDS, and then washing the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above-mentioned DNA molecules are also within the protection scope of the present invention.

The above-mentioned recombinant vector is a recombinant vector that inserts the above-mentioned DNA molecule into an expression vector to obtain the expression of the above-mentioned protein; specifically, inserts the nucleotides shown in the 10-2625th position from the 5' end of the sequence 2 in the sequence listing into the XbaI of the expression vector pCAMBIA2300 and the vector obtained between the SalI restriction site.

The application of the above-mentioned protein, the above-mentioned DNA molecule or the above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium in restoring the plant with the yellowing-to-greening trait to the plant with the greening trait is also within the protection scope of the present invention;

The yellowing-to-green trait is that the leaves of the plant are yellow-green in the 1-leaf stage-3 leaf stage, and the leaves turn green gradually from the 4-leaf stage, and the leaves are green in the 6th leaf stage-heading and fruiting stage;

The greening trait is that the leaves of the plant are all green in the 1-leaf stage-heading and fruiting stage;

Or the application of the above-mentioned protein, the above-mentioned DNA molecule, or the above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium in regulating the chlorophyll content of leaves of plants at the 1-leaf stage, 2-leaf stage, or 3-leaf stage;

The above regulation of plant chlorophyll content is to increase plant chlorophyll content.

The plant is specifically a dicotyledonous plant or a monocotyledonous plant, and the monocotyledonous plant is specifically rice, and the rice is especially japonica rice or indica rice.

The third object of the present invention is to provide a method for cultivating transgenic plants.

The method provided by the present invention is to introduce the DNA molecule encoding the above-mentioned protein into the target plant with the trait of yellowing and turning green, so as to obtain the transgenic plant with greening trait;

The chlorophyll content of the leaves of the transgenic plant at the 3-leaf stage is higher than that of the target plant;

The DNA molecule encoding the above-mentioned protein is specifically introduced into the target plant through the above-mentioned recombinant vector;

The plant is specifically a dicotyledonous plant or a monocotyledonous plant, and the monocotyledonous plant is specifically rice, and the rice is especially japonica rice or indica rice. The target plant is rice mutant gry79.

The yellow-to-green mutant gry79 (Oryza.sativa L.) in the present invention was preserved in the General Microorganism Center of China Committee for Microorganism Culture Collection (abbreviated as CGMCC, address: Beichen, Chaoyang District, Beijing) on January 14, 2014 No. 3, No. 1 Yard, West Road), the preservation number is CGMCC NO.8769, and the classification is named rice (Oryza.sativa).

The primer pair for amplifying the full length of the above-mentioned DNA molecule or any fragment thereof is also within the protection scope of the present invention.

The experiment of the present invention proves that using chemical mutagenesis to obtain the rice yellow-turning-green mutant gry79, comparing the wild type and the mutant, a new gene GRY79 was found in the wild type rice, and its mutant gene mGRY79 corresponding to the gene was found in the mutant , the mutant gene mGRY79 can be used as a leaf color marker gene for effective early identification of hybrid rice seedlings and efficient removal of false hybrids, thereby improving the field purity of hybrid rice. The new gene GRY79 was introduced into the rice yellow-turning-green mutant gry79 obtained by chemical mutagenesis, and the mutant returned to green color, and the chlorophyll content in the leaves increased, so this gene is related to chlorophyll synthesis and regulates leaf greenness.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The japonica rice line "lvp1"; the public can obtain it from Sichuan Agricultural University, and the non-patent documents that record the japonica rice line lvp1 are: Sun CH, Fang J, Zhao TL, Xu B, Zhang FT, Liu LC, Tang JY, Zhang GF, Deng XJ, Chen F, Qian Q, Cao XF, Chu CC(2012) The histone methyltransferase SDG724mediates H3K36me2/3deposition at MADS50and RFT1and promotes floweringin rice. Plant Cell24:3235-3247.

pCAMBIA2300; the public can obtain from Sichuan Agricultural University, and the non-patent documents that have recorded pCAMBIA2300 are: Ding Y, Wang X, Su L, Zhai JX, Cao SY, Zhang DF, Liu CY, Bi YP, Qian, Cheng ZK, ChuCC, Cao XF (2007) SDG714, a histone H3K9methyltransferase, is involved in Tos17DNAmethylation and transposition in rice. Plant Cell, 19:9-22. .

Agrobacterium EHA105; the public can obtain it from Sichuan Agricultural University, and the non-patent documents that have recorded Agrobacterium EHA105 are: Zhang ZH, Chen H, Huang XH, Xia R, Zhao QZ, Lai JB, Teng KL, Li Y, Liang LM, Du QS, Zhou XP, Guo HH, Xie Q. BSCTV C2 Attenuates the degradation of SAMDC1 to suppress DNA methylation-mediated gene silencing in Arabidopsis. Plant Cell, 2011, 23:273–288.

Example 1. Discovery and application of rice yellowing-turning-greening mutant gene mGRY79

1. Obtaining the yellow-to-green mutant gry79

The japonica rice line lvp1 (hereinafter also referred to as wild-type japonica rice) was treated with EMS mutagenesis, and a yellow-revertible-green mutant gry79 (green-revertible yellow79) that could be stably inherited was obtained in its offspring.

The leaves of the mutant were yellow-green at the 1st-3rd leaf stage, the chlorophyll content decreased, and the plant grew slowly. It gradually turned green from the 4th leaf stage, and the leaves were all green at the 6th leaf stage to the heading and fruiting stage.

From the above, it can be seen that the mutant has the trait of turning from yellow to green.

The yellow-to-green mutant gry79 (Oryza.sativa L.) was deposited in the General Microorganism Center of China Committee for Microbial Culture Collection (CGMCC for short) on January 14, 2014. The address is: Beichen West Road 1, Chaoyang District, Beijing No. 3), the preservation number is CGMCC NO.8769, and the classification is named rice (Oryza.sativa).

The trait of yellowing and turning green is that the leaves of the plant are yellow-green in the 1-3 leaf stage, and the leaves gradually turn green from the 4-leaf stage, and the leaves are green in the 6-leaf stage-heading and fruiting stage.

2. Discovery of the mutant gene mGRY79

Genetic analysis revealed that the yellow-to-green mutant gry79 is controlled by a pair of recessive nuclear genes. Through map-based cloning and sequencing, it was found that the gene LOC_Os02g33610 in the japonica rice line lvp1 and the yellow-turned-green mutant gry79 were different, and the gene LOC_Os02g33610 in the japonica rice line lvp1 was named GRY79.

GRY79 is located on the long arm of rice chromosome 2. The nucleotide sequence of its genomic DNA is the 10th-7501th position from the 5' end of the sequence 1 in the sequence listing, and its cDNA sequence is the 10th-7501th from the 5' end of the sequence 2 in the sequence listing. At position 2625, the lengths of the DNA and cDNA sequences are 7492bp and 2616bp respectively, and the encoded protein is named GRY79, and its amino acid sequence is shown in sequence 3 in the sequence listing. The amino acid sequence of protein GRY79 consists of 871 amino acid residues, and its molecular weight is about 96kD.

In the yellow-to-green mutant gry79, the GRY79 gene is mutated into the mGRY79 gene, and the sequence of the genomic DNA of mGRY79 is the sequence A from the 5' end to the 10th-7501st position, and the sequence A is the sequence 1 in the sequence table from the 5' end to the 4218th position The sequence obtained by mutating base C to T; the cDNA sequence of mGRY79 is sequence B from the 10th to 2625th position at the 5' end, and sequence B is the mutation of sequence 2 in the sequence table from the 1169th base C at the 5' end to T The amino acid sequence of the protein encoded by the mGRY79 gene is that only the 387th seramino (Ser) of sequence 3 in the sequence listing is changed to phenylalanine (Phe), and other amino acid residues are the same as sequence 3.

3. Application of the yellowing-turning-green mutant gry79 or the mutant gene mGRY79 in breeding rice male sterile lines with yellowing-turning-green traits

1) Cultivation of male sterile lines of rice with the trait of yellowing and turning green

According to conventional breeding methods, the yellowish-turned-green mutant gry79 can be crossed and/or backcrossed with a production hybrid rice sterile line (especially a photothermosensitive genic male sterile line for two-line hybrid rice) to breed a A new male sterile line with the trait of yellowing to greening.

2) Identify or assist in identifying whether the plant to be tested is a yellow-turned-green plant

Comparison of the yellow-to-green mutant gry79 with its wild-type japonica line lvp1:

By sequencing the genomic DNA, it can be seen that the only difference between the two at the gene level is the mGRY79 gene and the GRY79 gene;

By observing the leaves, it can be seen that the leaves of the yellow-to-green mutant gry79 are yellow-green in the 1-3 leaf stage, gradually turn green from the 4-leaf stage, and the leaves are all green in the 6-leaf stage-heading and fruiting stage; The leaves of the wild-type japonica rice line lvp1 are green throughout;

Phenotype of agronomic traits: At the maturity stage, compared with the wild type japonica rice lvp1, the yellow-to-green mutant gry79 had no significant difference in other main agronomic traits except that the number of effective panicles per plant decreased by 15%.

Therefore, the mutant gene mGRY79 in the yellowish-turned-green mutant gry79 can be used to identify or assist in identifying whether the plant to be tested is a yellowish-turned green plant; to detect the genomic DNA of the plant to be tested, if the genome of the plant to be tested contains the mutant gene mGRY79, the plant to be tested is or is a candidate for a yellowish-turned-green plant; if the genome of the tested plant does not contain the mutant gene mGRY79, the tested plant is not or a candidate is not a yellowish-turned-green plant.

Example 2. Acquisition and functional verification of the rice gene GRY79

1. Cloning of rice gene GRY79

Synthesize the following primer pairs:

P1: 5′CCG TCTAGA ATGGTGGCGCTCGCCTCC3′ (the underline is the XbaⅠ restriction site)

P2: 5′GCC GTCGAC TCATTGGGTTGCCATTTC3′ (the underline is the SalⅠ restriction site)

RNA was extracted from the leaves of the normal green japonica rice line lvp1, transcribed into cDNA by a reverse transcription kit, and amplified by RT-PCR with primers P1 and P2 to obtain a fragment of about 2500bp, which was ligated into the pMD-19-T vector, The obtained ligation product was transformed into Escherichia coli JM109 strain, and positive clones were screened to extract plasmids and sent for sequencing.

Sequencing results show that: the amplified fragment has the sequence shown in the 10-2625th nucleotide from the 5' end of the sequence 2 in the sequence listing, and the amplified fragment is the gene GRY79, and its open reading frame is the sequence 2 from the sequence listing. The amino acid sequence of the protein GRY79 encoded by the gene is sequence 3 in the sequence listing at positions 10-2625 of the 5' end.

The plasmid containing the fragment is named pMD-GRY79-1, which is a vector obtained by inserting the nucleotides shown in the 10th-2625th positions from the 5' end of sequence 2 in the sequence listing into the pMD-19-T vector.

2. Functional verification of the rice gene GRY79

1. Construction of expression vector

The plasmid pMD-GRY79-1 obtained in the above one was digested with XbaI and SalI to recover a 2616bp digested fragment, which was combined with the expression vector pCAMBIA2300 containing the act1 promoter from rice after the same double digested The backbone of 10379bp was connected to obtain a recombinant vector.

The recombinant vector was verified by XbaI and SalI double enzyme digestion, and the 2616bp was positive.

Then the positive recombinant vector was sent for sequencing. As a result, the recombinant vector was inserted into the XbaI and SalI restriction sites of the expression vector pCAMBIA2300 by inserting the nucleotides shown in the 10-2625th position from the 5' end of the sequence 2 in the sequence listing. The vector obtained between the points is a transgene recombinant expression vector named pCAMBIA2300-GRY79.

2. Obtaining of transgenic GRY79 rice

The recombinant vector pCAMBIA2300-GRY79 obtained in the above 1 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105/pCAMBIA2300-GRY79.

The plasmid of the recombinant Agrobacterium was extracted and sent for sequencing. The plasmid was pCAMBIA2300-GRY79, which proved that the recombinant Agrobacterium was positive.

The recombinant Agrobacterium EHA105/pCAMBIA2300-GRY79 was transferred into the rice yellow-turned-green mutant gry79 mature seeds to induce callus after shelling, and the recombinant Agrobacterium EHA105/pCAMBIA2300-GRY79 was co-cultured with the obtained callus for transformation. Then, through the steps of bacteria washing, screening, differentiation, rooting, seedling hardening, transplanting in field, etc., the transgenic GRY79 rice is obtained.

The leaf DNA of transgenic GRY79 rice was extracted from individual plants as a template for PCR amplification, and the primers were 5′-CAGGACTTACCTTGATGC-3′ and 5′-ACGACGGCCAGTGCCAAG-3′ (the GRY79 gene sequence and the vector respectively located in the aforementioned recombinant vector pCAMBIA2300-GRY79 The amplified product was detected by agarose gel electrophoresis, and the band with a size of 1918bp was positive for GRY79-transformed rice, and a total of 22 positive GRY79-transformed rice plants were obtained.

3. Phenotype study of transgenic GRY79 rice

The seeds harvested from positively transfected GRY79 rice, wild-type japonica rice lvp1 and mutant gry79 were sown and cultured under normal growth conditions. The experiment was repeated three times, with 20 seedlings for each positive transgenic GRY79 line and control materials (lvp1 and gry79), and the results were averaged.

1) Phenotype observation

The leaf phenotypes of positive transgenic GRY79 rice, wild-type japonica lvp1 and mutant gry79 were continuously observed.

Results The leaves of the mutant gry79 seedlings were yellow-green at the 1-3 leaf stage; while the leaves of the positive transgenic GRY79 rice seedlings recovered to the same color as the wild-type japonica rice lvp1 at the 1-3 leaf stage, all of which were green. The leaves of the positive transgenic GRY79 rice seedlings were also green from the 4-leaf stage to the heading and fruiting stage.

2) Detection of chlorophyll content

Take the leaves of positive transgenic GRY79 rice, wild-type japonica rice lvp1 and mutant gry79 seedlings at the three-leaf stage, weigh 0.2 g of each material with an electronic balance, cut it into pieces, and extract it with 80% acetone for 2 days at 4°C in the dark , transfer the extract into a 25mL volumetric flask to constant volume, and then use a UV-1700 (SHIMADZU) spectrophotometer to measure the absorbance at wavelengths of 663nm, 646nm and 470nm respectively, repeat 3 times for each sample, and take the average value to calculate the chlorophyll The content of chlorophyll, the non-patent literature that has recorded the specific method and calculation formula of the above-mentioned detection chlorophyll content is: Wang Pingrong, Wang Bing, Sun Xiaoqiu, Sun Changhui, Wan Chunmei, Ma Xiaozhi, Deng Xiaojian (2013) The fine location and physiological Characteristic Analysis. Chinese Agricultural Sciences, 46(2):225-232.

The result is as follows:

The chlorophyll content in leaves of mutant gry79 seedlings was 0.63 mg per gram of leaves (fresh weight);

The chlorophyll content in leaves of positively transfected GRY79 rice seedlings was 3.04 mg per gram of leaves (fresh weight);

The chlorophyll content in leaves of wild type japonica rice lvp1 was 3.34 mg per gram of leaves (fresh weight).

Chlorophyll content is chlorophyll a content + chlorophyll b content.

It can be seen that the chlorophyll content of positive transgenic GRY79 rice seedlings is also similar to that of the wild type at the three-leaf stage, indicating that the phenotype of the mutant has been restored.

Therefore, the GRY79 gene can restore the green color of rice leaves containing the mGRY79 mutant gene from the 1-leaf stage to the 3-leaf stage, and can increase the chlorophyll content.

Claims (10)

1. a DNA molecular is following 1) or 2) in any one DNA molecular:

1) coding region is for sequence A is from the DNA molecular shown in 5 ' end 10-7501 position, and described sequence A is that sequence in sequence table 1 is sported from 5 ' end the 4218th bit base C the sequence that T obtains;

2) coding region be sequence B from 5 ' end 10-2625 position, described sequence B is for sport T by sequence in sequence table 2 from 5 ' end the 1169th bit base C.

2. DNA molecular according to claim 1 has yellow and turns application in green proterties male sterible series of rice cultivating;

It is paddy rice is yellow-green colour at 1 leaf leaf phase-3, blade phase that described yellow turns green proterties, and from 4 leaf phases, blade turns green gradually, and the 6th leaf phase-heading and seeding stage blade is green.

3. application according to claim 2, is characterized in that: described paddy rice is japonica rice or long-grained nonglutinous rice.

4. whether DNA molecular described in claim 1 is the application that yellow turns in clear water rice assistant identification plant to be measured;

Describedly be applied as the genomic dna or cDNA that detect paddy rice to be measured, if containing in DNA molecular described in claim 11 in the genome of paddy rice to be measured) shown in DNA molecular or paddy rice to be measured cDNA in containing in DNA molecular described in claim 1 2) shown in DNA molecular, then paddy rice to be measured for or candidate turn clear water rice for yellow, if containing in DNA molecular described in claim 11 in the genome of paddy rice to be measured) shown in DNA molecular or paddy rice to be measured cDNA in containing in DNA molecular described in claim 1 2) shown in DNA molecular, then paddy rice to be measured not for or candidate not for yellow turns clear water rice,

It is paddy rice is yellow-green colour at 1 leaf leaf phase-3, blade phase that described yellow turns green proterties, and from 4 leaf phases, blade turns green gradually, and the 6th leaf phase-heading and seeding stage blade is green.

5. application according to claim 4, is characterized in that: described paddy rice is japonica rice or long-grained nonglutinous rice.

6. albumen, DNA molecular or the application that to revert in the paddy rice making to have yellow and turn green proterties containing the recombinant vectors of described DNA molecular, expression cassette or recombinant bacterium in the paddy rice with greening proterties;

Described albumen, the protein be made up of the aminoacid sequence shown in sequence in sequence table 3;

Described DNA molecular is following 1) or 2) in any one DNA molecular:

1) coding region is for sequence in sequence table 2 is from the DNA molecular shown in the DNA molecular shown in 5 ' end 10-2625 position Nucleotide;

2) coding region is for sequence in sequence table 1 is from the DNA molecular shown in 5 ' end 10-7501 position Nucleotide;

It is paddy rice is yellow-green colour at 1 leaf leaf phase-3, blade phase that described yellow turns green proterties, and from 4 leaf phases, blade turns green gradually, and the 6th leaf phase-heading and seeding stage blade is green;

Described greening proterties be paddy rice 1 leaf phase-heading and seeding stage blade is green.

7. albumen, DNA molecular or the recombinant vectors containing described DNA molecular, expression cassette or recombinant bacterium are in the application of adjusting and controlling rice in 1 leaf phase or 2 leaf phases or 3 leaf phase chlorophyll content in leaf blades;

Described albumen, the protein be made up of the aminoacid sequence shown in sequence in sequence table 3;

Described DNA molecular is following 1) or 2) in any one DNA molecular:

1) coding region is for sequence in sequence table 2 is from the DNA molecular shown in the DNA molecular shown in 5 ' end 10-2625 position Nucleotide;

2) coding region is for sequence in sequence table 1 is from the DNA molecular shown in 5 ' end 10-7501 position Nucleotide.

8. application according to claim 7: described paddy rice is japonica rice or long-grained nonglutinous rice.

9. cultivate a method for transgenic plant, turn in the object paddy rice of green proterties for the importing of the DNA molecular of proteins encoded is had yellow, obtain the transgenic paddy rice with greening proterties;

Described albumen, the protein be made up of the aminoacid sequence shown in sequence in sequence table 3;

Described DNA molecular is following 1) or 2) in any one DNA molecular:

1) coding region is for sequence in sequence table 2 is from the DNA molecular shown in the DNA molecular shown in 5 ' end 10-2625 position Nucleotide;

2) coding region is for sequence in sequence table 1 is from the DNA molecular shown in 5 ' end 10-7501 position Nucleotide;

Described transgenic plant at the chlorophyll content of 3 leaf phase blades higher than described object paddy rice;

The DNA molecular of described proteins encoded is imported in described object paddy rice by the recombinant vectors containing described DNA.

10. method according to claim 9, is characterized in that: described paddy rice is japonica rice or long-grained nonglutinous rice.