CN110184281B - Rice seed storability gene OsGH3-2 and application of molecular marker thereof - Google Patents

Abstract

The invention provides a rice seed storability gene OsGH3-2 and the application of its molecular marker. The present invention clones the rice seed storability gene OsGH3-2, and its nucleotide sequence is as shown in SEQ ID NO.1 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The present invention verifies the function of the gene in improving the storage tolerance of rice seeds through genetic transformation: using RNAi technology to induce the loss of OsGH3-2 gene function and RNAi to reduce the expression of the OsGH3-2 gene, confirming that the gene can significantly enhance rice seeds storability. The present invention also provides a molecular marker closely linked with the OsGH3-2 gene, which can be used to detect the OsGH3-2 haplotype and gene selection, and realize its application in cultivating rice varieties with strong seed storage tolerance.

Description

A rice seed storage tolerance gene OsGH3-2 and its application of molecular markers

technical field

The invention belongs to the field of crop molecular biology, in particular to the application of a rice seed storage tolerance gene OsGH3-2 and its molecular marker.

Background technique

Seed is the carrier of plant genetic material, and it is also the basic material information and main output form of agricultural production. After harvesting, crop seeds are inevitably aged by storage environment, resulting in a decline in viability, which in turn affects their seed value and crop yield (Yamauchi and Winn 1996). Due to the arrangement of crop seasons, the market behavior of seed supply and the policy needs of stabilizing agricultural production, a certain amount of seed reserves or stocks are required all year round. In recent years, the average seed inventory of major crops such as rice, corn, and wheat in my country is close to 1.2 billion kilograms. The decline in viability during storage not only leads to huge commercial losses, but also increases the risk of grain production (Gao Liwei et al. 2016, National Bureau of Statistics 2017). Seed storability refers to the ability of seeds to maintain seed vigor after long-term storage. Seeds with strong storability can still have high viability and sowing value after a period of storage, extending the timeliness of commercial seed supply. Therefore, seed storability is an important agronomic trait and commodity characteristic, which is directly related to the effect of seed production and utilization. Seeds undergo enhanced metabolism induced by the storage environment, and excessive accumulation of reactive oxygen species induces damage to biomacromolecules, such as lipid membrane peroxidation, protein carbonylation, and nucleic acid strand breaks, resulting in seed deterioration (Parkhey et al 2012). Plant self-protection mechanism and oxidative damage repair system based on inhibiting the production of reactive oxygen species or enhancing the scavenging ability are the keys to regulating the storability of seeds. Rice is an important food crop, and the humid and hot environment in its production area easily induces seed deterioration, resulting in a shorter seed life, generally only 1-2 years. Therefore, improving the storability of rice seeds can prolong the planting time, improve the quality of seeds, and retain the original processing and nutritional quality of the grain, which has great social and economic value for ensuring the security of food supply (Hu Jin 2014).

Seed storability is a complex quantitative trait controlled by multiple genes (Bentsink et al 2006). There are wide variations in the storability of rice seeds. Generally, wild rice is stronger than indica rice, and indica rice is stronger than japonica rice (Zeng Dali et al. 2002, Wu Fangxi et al. 2010). At present, researchers have detected more than 50 QTLs related to seed storability using multiple rice genetic populations (Miura et al 2002, Zeng et al 2006, Li et al 2012, Hang et al 2014, Dong et al2017, Jinet al 2018, Liu et al. et al 2018). However, the storability gene mining of rice seeds has progressed slowly, and there is no report on the cloning of storability genes in rice seeds using natural allele variation (Kretzschmar et al 2015). Therefore, using forward genetics to mine seed storability genes is the most economical and effective means to reduce seed storage costs and improve seed quality.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a rice seed storage tolerance gene OsGH3-2 and its molecular marker and application.

The present invention clones the rice seed storage tolerance gene OsGH3-2, and confirms that the gene negatively regulates the seed storage tolerance through genetic transformation. Through the analysis of allele function and haplotype function, it is confirmed that the gene promoter variation-446 SNP leads to gene function variation, and the present invention also develops a molecular marker for detecting the genotype of this site.

The nucleotide sequence of the rice seed storage tolerance gene OsGH3-2 provided by the present invention is shown in SEQ ID No. 1, or the nucleotide sequence shown in SEQ ID No. 1 is substituted, deleted and/or added by one or more genes encoding the same functional protein.

The present invention also provides the protein encoded by the rice seed storage tolerance gene OsGH3-2, which has:

1) the amino acid sequence shown in SEQ ID No.2; or

2) A protein derived from 2) in which the amino acid sequence shown in SEQ ID No. 2 is substituted, deleted and/or added by one or more amino acids and has equivalent activity.

The present invention provides a biological material containing the OsGH3-2 gene, and the biological material is a vector, a transgenic cell line, an engineered bacteria, a host cell or an expression cassette.

The present invention provides the application of the above-mentioned rice seed storability gene OsGH3-2, or the protein encoded by it, or a biological material containing the gene, in improving the storability of plant seeds.

The present invention provides the application of the above-mentioned rice seed storage tolerance gene OsGH3-2, or the protein encoded by it, or the biological material containing the gene, in improving the germination rate of plant seeds after storage.

The invention provides the application of the above-mentioned rice seed storage tolerance gene OsGH3-2, or the protein encoded by it, or the biological material containing the gene, in prolonging the planting time of plant seeds and improving the quality of seeds.

The present invention confirms that the gene OsGH3-2 is a negative regulation gene of rice seed storability. Based on the findings of the present invention, those skilled in the art can understand that a method known in the art for knocking out the OsGH3-2 gene, such as CRISPR-Gas9 technology, or methods to reduce the expression of OsGH3-2 gene, such as RNAi method, to prevent or reduce the expression of OsGH3-2 gene, can improve the storage tolerance of plant seeds, improve the germination rate of plant seeds after storage, and prolong the use of plant seeds. time and the purpose of improving seed quality. Therefore, those skilled in the art can understand that the above applications are all negative regulation applications of the OsGH3-2 gene.

Based on this, the application provides an expression inhibitor of the OsGH3-2 gene or any of the following applications of the method for inhibiting the expression of the gene:

(1) application in improving the storage resistance of plant seeds;

(2) application in improving the germination rate of plant seeds after storage;

(3) The application of prolonging the planting time of plant seeds and improving the quality of seeds.

The present invention provides the application of the above-mentioned rice seed storability gene OsGH3-2, or its encoded protein, or a biological material containing the gene in plant breeding, germplasm resource improvement, or cultivation of transgenic plants with strong seed storability.

In the above application, the plants are rice, soybean, wheat, barley, sorghum, millet, sesame, rape, corn, peanut. Preferably, the plant is rice.

The present invention also provides a molecular marker of the rice seed storage tolerance gene OsGH3-2, which is the OsGH3-2 promoter-446 base SNP variation, which is located at the 155th position of the nucleotide sequence shown in SEQ ID No.3 , and its polymorphism is A/G.

Further, the molecular marker described in the present invention can be amplified by the following primer combination, and the primer combination contains 3 primers whose nucleotide sequences are:

Upstream primer 1: CTTATTAATTTCTCCATCAGATGAGAA (SEQ ID NO. 4)

Upstream primer 2: CTTATTAATTTCTCCATCAGATGAGAG (SEQ ID NO. 5)

Universal primer: CGTCTCCGGGATTAATCAACGGC (SEQ ID NO. 6).

The present invention also provides a set of primer combinations for detecting the storability of rice seeds, comprising three primers whose nucleotide sequences are:

Upstream primer 1: CTTATTAATTTCTCCATCAGATGAGAA (SEQ ID NO. 4)

Upstream primer 2: CTTATTAATTTCTCCATCAGATGAGAG (SEQ ID NO. 5)

Universal primer: CGTCTCCGGGATTAATCAACGGC (SEQ ID NO. 6).

The present invention provides any of the following applications of the above-mentioned molecular marker or primer combination:

(1) Screening rice seeds with strong storability;

(2) Eliminate rice seeds with weak storability;

(3) Detecting rice seeds with high germination rate or rice seeds with low germination rate during the storage process of rice seeds;

(4) Screening of rice seeds with long seeding time and high quality;

(5) Cultivate rice varieties with strong storage tolerance.

Preferably, in the above application, the primers shown in SEQ ID NO. 4-6 are used to detect the PCR amplification product based on the KASP reaction system platform, and the judgment is made according to the fluorescence signal of the amplification product.

The present invention provides a method for detecting the above-mentioned molecular markers. The following primer combinations are used to amplify the genomic DNA of the rice to be tested, and the PCR amplification products are detected based on the KASP reaction system platform:

The nucleotide sequence of the primer combination is:

Upstream primer 1: CTTATTAATTTCTCCATCAGATGAGAA (SEQ ID NO. 4)

Upstream primer 2: CTTATTAATTTCTCCATCAGATGAGAG (SEQ ID NO. 5)

Universal primer: CGTTCCGGGATTAATCAACGGC (SEQ ID NO. 6);

If only the fluorescent signal corresponding to upstream primer 1 is detected in the sample PCR product, the SNP site to be detected is base A, and it is determined that the test sample contains OsGH3-2 haplotype 4, and the seeds containing this allele have poor storage resistance. ; If only the fluorescence signal corresponding to the upstream primer 2 is detected, the SNP site to be detected is the base G, that is, the test sample does not contain OsGH3-2 haplotype 4, and the seeds containing this allele have strong storage resistance; If two fluorescent signals are detected at the same time, the detection site is A:G, which contains the heterozygous OsGH3-2 gene.

The present invention clones the rice seed storage tolerance gene OsGH3-2, and verifies the function of the gene through genetic transformation: using RNAi technology to induce the loss of OsGH3-2 gene function and RNAi to reduce the expression of the OsGH3-2 gene, confirming the gene It can significantly enhance the storability of rice seeds. The invention also provides a molecular marker closely linked with the OsGH3-2 gene, which can be used to detect the OsGH3-2 allele and gene selection, and realize its application in breeding rice varieties with strong seed storage tolerance.

Description of drawings

Figure 1 is a schematic diagram of qSS1 near-isogenic line genotype and seed storage tolerance phenotype.

Figure 2 is a schematic diagram of qSS1 fine mapping and sequence variation in candidate gene regions.

Figure 3 is a graph showing the results of OsGH3-2 overexpression and storability phenotype of RNAi transgenic seeds. OX5, 6, 7 respectively represent OsGH3-2 overexpression positive families; RNAi1, 2, 3 represent OsGH3-2 RNAi positive families, respectively.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention.

Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art; unless otherwise specified, the reagents used in the examples are all commercially available.

Example 1 Acquisition and phenotypic analysis of rice seed storage tolerance gene OsGH3-2

1. Excavation of storability loci in rice seeds

The present invention uses the backcross recombinant inbred line population constructed by NIP and 9311 to detect four storability QTLs, among which qSS1 has the greatest genetic effect, and the candidate gene region is located on chromosome 1 31,849,525-32,274,465. Through molecular marker-assisted selection, a near-isogenic line NIL-qSS1 ^NIP with 9311 genotype replaced by qSS1 and 9311 genotype in the rest of the genome was obtained. After natural aging and artificial aging (achieved by the accelerated deterioration of seeds induced by high temperature and high humidity, this example is to accelerate the aging of seeds in an environment of 43 ° C and a humidity of 90%), the germination rates of the two seeds have the same trend, NIL-qSS1 The germination rates of ^NIP seeds were significantly lower than those of 9311 (B panel in Figure 1 ), that is, the 9311 allele of qSS1 enhanced the storability of seeds.

2.qSS1 fine positioning

The flanking markers RM11698 and RM11716 were used to genotype 6048 isolates constructed by backcrossing NIL-qSS1 ^NIP with 9311, and a total of 145 recombinant individuals were obtained (Figure 2A). The recombinant single species was planted into a family, the single plant was harvested, and the storability phenotype of the seeds was identified by artificial aging. Combined with the genotype of the recombinant family and the analysis of seed storability, qSS1 was finally fine-mapped to the 4.4kb region between RM11709 and Indel2 (Figure 2, panel B). The candidate gene region contains only one annotated gene, LOC_Os01g55940, which encodes an IAA amide synthase, named OsGH3-2. Through sequence alignment, it was found that NIP and 9311 in the candidate gene region contained only 4 SNPs in the promoter region and only 1 non-synonymous mutation in the gene coding region (C panel in Figure 2). The expression levels of OsGH3-2 in leaves of 9311 and NIL-qSS1 ^NIP were detected by Realtime-PCR, and it was found that the expression level of OsGH3-2 in 9311 was significantly lower than that of NIL-qSS1 ^NIP (D panel in Figure 2). The near-isogenic lines showed that the higher the expression of OsGH3-2 allele, the worse the storability of seeds. Therefore, it is speculated that this gene negatively regulates the storability of seeds.

Table 1 Polymorphic molecular markers closely linked to qSS1

3. Functional analysis of OsGH3-2

In order to verify the gene function of OsGH3-2, the present invention cloned the OsGH3-2 gene coding region 3221bp from the 9311 genome, constructed it into the pCambia1301 vector, and overexpressed it driven by the 35S promoter. The above-mentioned overexpression vector was transformed into Zhonghua 11, and 56 positive transgenic plants of T0 generation were obtained. Select 3 positive T0 generation single plants with significantly increased expression levels to plant and harvest the T1 generation family seeds (Figure 3A). After natural aging (one year at room temperature), the seed germination rate was investigated, and it was found that the seed germination rate of the three independent T1-positive families was significantly reduced (Table 2), that is, the seed storage tolerance was significantly weaker than that of the wild type (Fig. 3B).

Table 2 Seed germination rate after artificial aging of OsGH3-2 overexpressed transgenic materials

In addition, the present invention selects the 3'513bp of the OsGH3-2 coding region as the RNAi target site to construct the RNA interference genetic transformation material. Three T0-generation positive families with significantly reduced OsGH3-2 expression were selected to investigate the seed storage tolerance phenotype (C in Figure 3). After artificial aging, the seed germination rate of OsGH3-2 RNAi-positive families was significantly higher than that of wild-type Zhonghua 11, as shown in Table 3, indicating that OsGH3-2 negatively regulates the storability of rice seeds. Breeders can select for OsGH3-2 non-haplotype 4 alleles to enhance the storability of rice seeds.

Table 3 Seed germination rate after artificial aging of OsGH3-2RNAi transgenic materials

Example 2 Acquisition of OsGH3-2 molecular marker

Haplotype analysis of OsGH3-2 was carried out by using SNPs in the fine mapping region of qSS1, and it was found that OsGH3-2 mainly had 4 haplotypes in the rice core germplasm, and the storage tolerance of seeds of the 4 haplotypes was significantly lower than that of the other 3 haplotypes. haplotype (Table 2), thus it is judged that SNP-446 causes a functional variation of the OsGH3-2 allele, and the variation occurs at position 155 in SEQ ID NO.3.

Table 4 OsGH3-2 haplotype analysis results

Note: The same letter as ab indicates that there is no significant difference in rice seed longevity after multiple comparisons (LSD Duncan).

In the present invention, the single base variation of SNP-446 is developed into KASP (Kompetitive Allele Specific PCR) marker for gene selection. The sequence of the developed marker K_OsGH3-2-446 is as follows:

Upstream primer 1: CTTATTAATTTCTCCATCAGATGAGAA (SEQ ID NO. 4)

Upstream primer 2: CTTATTAATTTCTCCATCAGATGAGAG (SEQ ID NO. 5)

Universal primer: CGTCTCCGGGATTAATCAACGGC (SEQ ID NO. 6)

The 5' of the two specific primers were respectively connected to the FAM or HEX linker sequences of LGC Company. The KASP reaction system refers to the instructions of the KASP Master Mix reagent, and the matching LGC SNP line genotyping platform is used for the result detection.

If only the fluorescent signal corresponding to upstream primer 1 is detected in the sample PCR product, the detection site is base A, and it is determined that the test sample contains OsGH3-2 haplotype 4, that is, the allele seed has poor storage resistance; If the fluorescence signal corresponding to the upstream primer 2 is detected, the detection site is base G, that is, the OsGH3-2 in the test sample is not haplotype 4, that is, the allele seeds have strong storage resistance; If the fluorescent signal is detected, the site is A:G, and it contains the heterozygous OsGH3-2 gene.

Twenty-three copies of rice OsGH3-2 gene promoter-446 nucleotide were detected by marker K_OsGH3-2-446. The analysis results of the marker K_OsGH3-2-446 are shown in Table 5, which are completely consistent with the sequencing results of this site, indicating that the marker can accurately detect the OsGH3-2 haplotype in rice materials. In addition, the analysis of K_OsGH3-2-446 showed that the storability of the rice material with the G locus was generally stronger than that of the rice material with the locus A, so it can be judged that this marker can not only analyze the OsGH3-2 allele genotype, but also The storability of material seeds can also be predicted.

Table 5 Test genotyping data for marker K_OsGH3-2-446

Taking the seed life as an indicator, the longer the seed life, the stronger the storability. The marker can not only accurately distinguish locus genotypes, but also better judge the storability of rice seeds.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principles of the present invention, several improvements and modifications can be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

sequence listing

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<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 24

agaagggctg aatctctcca 20

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 25

gtctgagaaa cgtggttcca c 21

<210> 26

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 26

cacttgactg tgcaagagat g 21

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 27

gtccatcacg acgaaccaac 20

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 28

aacccctgtc aaaaccatcc 20

<210> 29

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 29

actggagaag aaaggccgaa 20

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 30

gtcttgcatg cttgtggagt 20

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 31

atgaattgtg tcgtcggcag 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 32

acatcgctga gttttgaggc 20

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 33

gtgattgcga agtcatgcgt 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 34

ccgccactac acaaacacat 20

<210> 35

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 35

gaactaatca agcatgcacg ag 22

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 36

tttcatctca agtttgttca cgt 23

<210> 37

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 37

gctatttccc attccaggcc 20

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 38

ctgggtttga ggttgtgtcg 20

Claims (8)

1. The application of rice seed storability gene OsGH3-2, or the protein encoded by it, or a biological material containing the gene in improving the storability of rice seeds, The nucleotide sequence of the rice seed storage tolerance gene OsGH3-2 is shown in SEQ ID No.1; The amino acid sequence of the encoded protein is shown in SEQ ID No.2; The biological material is a vector, host cell or expression cassette. 2. Application of the rice seed storability gene OsGH3-2, or the protein encoded by it, or the biological material containing the gene in improving the germination rate of rice seeds after storage, the core of the rice seed storability gene OsGH3-2 The nucleotide sequence is shown in SEQID No.1; The amino acid sequence of the encoded protein is shown in SEQ ID No.2; The biological material is a vector, host cell or expression cassette. 3. The application of rice seed storability gene OsGH3-2, or the protein encoded by it, or the biological material containing the gene, in prolonging the planting time of rice seeds, the nucleoside of the rice seed storability gene OsGH3-2 The acid sequence is shown in SEQ IDNo.1; The amino acid sequence of the encoded protein is shown in SEQ ID No.2; The biological material is a vector, host cell or expression cassette. 4. Application of the rice seed storability gene OsGH3-2, or the protein encoded by it, or the biological material containing the gene in cultivating transgenic rice with strong seed storability, the rice seed storability gene OsGH3-2 The nucleotide sequence is shown in SEQ ID No.1; The amino acid sequence of the encoded protein is shown in SEQ ID No.2; The biological material is a vector, host cell or expression cassette. 5. The molecular marker of rice seed storability gene OsGH3-2, characterized in that, the molecular marker is as shown in SEQ ID No. 3, and A/G mutation occurs at position 155 of the sequence, resulting in more storability of rice seeds. attitude. 6. A set of primer combinations for detecting the storability of rice seeds, characterized in that it contains 3 primers, the nucleotide sequences of which are respectively: Upstream primer 1: CTTATTAATTTCTCCATCAGATGAGAA (SEQ ID NO. 4) Upstream primer 2: CTTATTAATTTCTCCATCAGATGAGAG (SEQ ID NO. 5) Universal primer: CGTCTCCGGGATTAATCAACGGC (SEQ ID NO. 6). 7. the following any application of the molecular marker described in claim 5 or the primer combination described in claim 6: (1) Screening rice seeds with strong storability; (2) Eliminate rice seeds with weak storability; (3) Detecting rice seeds with high germination rate or rice seeds with low germination rate during the storage process of rice seeds; (4) Screening rice seeds with long seeding time; (5) Cultivate rice varieties with strong storage tolerance. 8. The method for detecting the storability of rice seeds, characterized in that the following primer combinations are used to amplify the genomic DNA of the rice to be tested, and the PCR amplification product is detected based on the KASP reaction system platform: The nucleotide sequence of the primer combination is: Upstream primer 1: CTTATTAATTTCTCCATCAGATGAGAA (SEQ ID NO. 4) Upstream primer 2: CTTATTAATTTCTCCATCAGATGAGAG (SEQ ID NO. 5) Universal primer: CGTCTCCGGGATTAATCAACGGC (SEQ ID NO. 6); If only the fluorescent signal corresponding to upstream primer 1 is detected in the sample PCR product, the SNP site to be detected is base A, and the seeds containing this allele have poor storage resistance; if only the fluorescent signal corresponding to upstream primer 2 is detected , the SNP site to be detected is the base G, and the seeds containing this allele have strong storage resistance; if two kinds of fluorescent signals are detected at the same time, the detection site is A:G, which contains the heterozygous OsGH3-2 Gene.

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