Application of rice OsGLP3-7 gene in regulating drought tolerance of rice seedling stage

Abstract

The application belongs to the technical field of crop molecular breeding, and particularly relates to application of rice OsGLP3-7 gene in regulation of drought tolerance of rice seedling stage. The application proves for the first time that the rice gene OsGLP3-7 is a functional gene for regulating drought tolerance of rice seedling stage, and cloning and biological function verification of the gene have important reference significance for the study of molecular mechanism of drought tolerance of rice seedling stage. The characteristics of the OsGLP3-7 gene and the protein coded by the gene in regulation of drought tolerance of rice seedling stage are utilized, and a genome targeting modification technology such as CRISPR / Cas9 is adopted to screen the strain with improved drought tolerance of rice seedling stage by mutating the nucleotide sequence of the gene, and the application has very important application in agricultural production. It is indicated that the rice OsGLP3-7 gene as a breakthrough point can provide an efficient breeding mode for creating rice varieties, germplasm resources and hybrid rice parents with enhanced drought tolerance of seedling stage.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop molecular breeding, and particularly relates to application of a rice OsGLP3-7 gene in regulating drought tolerance of rice seedlings. BACKGROUND

[0002] Rice, as the staple food of more than half of the world's population, occupies an irreplaceable position in the global food system. In recent years, the global population continues to grow rapidly, and the population will continue to rise in the next few decades. In order to ensure the food supply worldwide, the global rice production needs to increase by at least 1.0%-1.2% per year. However, the production of rice is facing severe challenges, and various adverse environmental stresses such as drought frequently affect rice yield.

[0003] Rice is extremely sensitive to drought stress, and its growth will be greatly inhibited once it encounters drought. According to research, even moderate drought stress will cause rice yield to decrease by more than 45%. Under the influence of human activities, the global climate continues to warm, and the trend of drought on earth is becoming more and more obvious. According to the climate model prediction, by 2050, more than 50% of the earth's area will be subjected to drought stress, which undoubtedly brings great crisis to rice production.

[0004] Under this situation, breeding drought-tolerant rice varieties becomes the most economical and effective way to solve this problem. It not only can reduce the impact of drought on rice yield, but also can reduce irrigation water, which is in line with the concept of sustainable development, and lays a solid foundation for global food security. Therefore, it is of great significance to reveal the molecular mechanism of drought tolerance of rice, to excavate important genes that regulate drought tolerance of rice, and to genetically improve drought tolerance of rice for the purpose of ensuring global food security.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the present application uses CRISPR / Cas9 technology to construct an OsGLP3-7 gene editing rice transformation vector, and carries out transformation experiments. Through analysis of the drought tolerance phenotype of transgenic plants, the function of the rice gene OsGLP3-7 as a regulatory gene for drought tolerance of rice seedlings is verified. After knocking out the OsGLP3- gene and destroying the biological function of the OsGLP3-7 gene, a seedling drought tolerance improved strain can be obtained. The above research results not only deepen the understanding of the molecular mechanism of drought tolerance of rice, but also provide gene resources and technical support for molecular breeding of new drought-tolerant rice varieties.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0008] The application provides the application of the rice OsGLP3-7 gene in regulating drought tolerance of rice seedlings, and the nucleotide sequence of the OsGLP3-7 gene is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0009] It should be understood that, considering the degeneracy of codons, the modification of the nucleotide sequence of the above-mentioned coding gene without changing the amino acid sequence also belongs to the protection scope of the application.

[0010] Preferably, the method for regulating is that the drought tolerance of rice seedlings is improved after the OsGLP3-7 gene is knocked out and the biological function of the OsGLP3-7 gene is destroyed.

[0011] The application provides a molecular breeding method for improving the drought tolerance of rice seedlings, i.e., the OsGLP3-7 gene in rice is knocked out by a CRISPR / Cas9 editing system to destroy the biological function of the OsGLP3-7 gene, and then a rice strain with improved drought tolerance in the seedling stage is obtained.

[0012] Preferably, the OsGLP3-7 gene editing vector is constructed by using the CRISPR / Cas9 technology, and the rice is transformed to knock out the OsGLP3-7 gene in the rice, destroy the biological function of the OsGLP3-7 gene, and then the rice strain with improved drought tolerance in the seedling stage is obtained after screening of offspring.

[0013] More preferably, the molecular breeding method for improving the drought tolerance of rice seedlings specifically comprises the following steps:

[0014] S1, editing vector construction: according to the CRISPR / Cas9 editing technology, two target sequences are selected in the coding region of OsGLP3-7, then two sgRNA expression cassettes containing the target sequence of the coding region of OsGLP3-7 are cloned into the binary vector pYLCRISPR / Cas9Pubi-H, so as to form a gene editing vector;

[0015] S2, the editing vector is transformed into the rice plant by using the agrobacterium-mediated genetic transformation method;

[0016] S3, referring to the sequence of the OsGLP3-7 gene of the receptor variety, a homozygous mutant strain of the OsGLP3-7 gene is screened, and then the mutant strain is propagated to obtain a strain with improved drought tolerance in the seedling stage.

[0017] Further, the two target sequences designed in the coding region of the OsGLP3-7 gene are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0018] Further, two sgRNA expression cassettes are constructed by repeated superposition method using primers OsGLP3-7-6aF / OsGLP3-7-6aR and OsGLP3-7-6bF / OsGLP3-7-6bR; the sequence of OsGLP3-7-6aF is shown as SEQ ID NO. 5, the sequence of OsGLP3-7-6aR is shown as SEQ ID NO. 6, the sequence of OsGLP3-7-6bF is shown as SEQ ID NO. 7, and the sequence of OsGLP3-7-6bR is shown as SEQ ID NO. 8.

[0019] Further, the rice plant uses Nipponbare rice variety.

[0020] Further, the OsGLP3-7 gene mutant strain is screened using primers OsGLP3-7JC-F / OsGLP3-7JC-R, wherein the sequence of OsGLP3-7JC-F is shown as SEQ ID NO. 19, and the sequence of OsGLP3-7JC-R is shown as SEQ ID NO. 20.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application first proves that the rice gene OsGLP3-7 is a functional gene for regulating drought tolerance of rice seedlings, and the cloning and biological function verification of the gene have important reference significance for the molecular mechanism research of drought tolerance of rice seedlings. Meanwhile, the present application uses the characteristics that the OsGLP3-7 gene and the protein encoded thereby are involved in the regulation of drought tolerance of rice seedlings, and adopts CRISPR / Cas9 and other genome targeting modification technologies to screen rice seedling drought tolerance improved strains by mutating the nucleotide sequence of the gene, which has very important application in agricultural production. In addition, the present application takes the rice OsGLP3-7 gene as a starting point to provide an efficient breeding method for creating rice varieties, germplasm resources, and hybrid rice parents with enhanced seedling drought tolerance, and realizes the precise improvement of the seedling drought tolerance of rice by developing and utilizing the OsGLP3-7 gene. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Gene editing of OsGLP3-7 knockout homozygous strain (Nip represents wild type Nipponbare);

[0024] Figure 2 Knocking out OsGLP3-7 significantly improves the seedling drought tolerance of rice plants. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application will be further described below. It should be noted that the description of these embodiments is intended to help understand the present application and is not intended to limit the present application. Moreover, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0027] Example: Study on the Regulation of Rice OsGLP3-7 Gene on Drought Tolerance of Rice Seedling Stage

[0028] Rice gene OsGLP3-7, whose nucleotide sequence is shown as SEQ ID NO. 1 and whose amino acid sequence is shown as SEQ ID NO. 2, is derived from japonica variety Nipponbare.

[0029] Nucleotide sequence of OsGLP3-7 (SEQ ID NO. 1):

[0030] ATGTCCTCATCATCATCAATGGAGTGCACCGGAAACATGTCGGCGGCGCCATTGCTCGTCTTGACGGTGGCGGTGCTCGCCGTGCTAGCCTCCACCTGCGCCGCCGACCCAGAGCCGATACAAGACTTCTGCGTGGCCGTGCCCCGTGCCGGCGGCGAGGCGTCGCCGGCATACCCGGGCTTCCCGTGCAAGCCGGCGTCCGCGGTCGTCTCCGACGACTTCTTCTTCGCCGGGCTCGCCGCCGCCGGCAGCACGGACAACCCGTTCGGGGCCAGCCTGAAGCCGGGCAACGTGGAGGCGTTCCCGGCGCTGAACACGCTCGGCGTCGCCATCAACCGCGTCGACCTCGCCCCCGGCGGCGTCAACCCGCTGCACAGCCACCCGCGCGCCGCCGAGCTGGTGCACGTCATCACCGGCCGGATGCTCGTCGGGTTCGTGAGCACGGCGGGGAAGTACTACTCCAAGGTGGTCGGCGAGGGGGAGACGTTCGCCATCCCGCGCGGGCTGATGCACTTCCAGTACAACCCTGGGAACGCCTCCGCCCGCGCCATGACGGTGTTCAACAGCCAGCTCCCCGGCGTCGTGCCCGCCGCGACGGCGCTGTTCGGCGCCGACCCGGAGATCCCCGACGCCGTCCTCGCCAAGAGCTTCCAGGTGGACGCCGAGATCATCAAACTGCTCAAGTCCAAGTTCAAGAAGTGA.

[0031] Amino acid sequence of OsGLP3-7 (SEQ ID NO. 2):

[0032] MSSSSSMECTGNMSAAPLLVLTVAVLAVLASTCAADPEPIQDFCVAVPRAGGEASPAYPGFPCKPASAVVSDDFFFAGLAAAGSTDNPFGASLKPGNVEAFPALNTLGVAINRVDLAPGGVNPLHSHPRAAELVHVITGRMLVGFVSTAGKYYSKVVGEGETFAIPRGLMHFQYNPGNASARAMTVFNSQLPGVVPAATALFGADPEIPDAVLAKSFQVDAEIIKLLKSKFKK.

[0033] 1. Construction of OsGLP3-7 gene editing vector

[0034] The gene sequence of OsGLP3-7 was obtained from the Rice Genome Annotation Project (http: / / rice.uga.edu / ) database, and two target points "TCATCATCAATGGAGTGCAC (SEQ ID NO. 3)" and "GCAGCACGGACAACCCGTTC (SEQ ID NO. 4)" were designed in the coding region of OsGLP3-7 gene according to the CRISPR / Cas9 editing technology, and primers OsGLP3-7-6aF / OsGLP3-7-6aR and OsGLP3-7-6bF / OsGLP3-7-6bR were synthesized according to the target sequence. Then the sgRNA expression cassette was constructed by repeated overlapping method (Overlapping PCR). The U6a and U6b promoters were amplified by the first round of PCR with the adapter primers U-F / OsGLP3-7-6aF and U-F / OsGLP3-7-6bF, respectively; the U6a and U6b terminators were amplified by the adapter primers gR-R / OsGLP3-7-6aR and gR-R / OsGLP3-7-6bR, respectively. After the first round of PCR products were diluted 10 times, 1 μL of each was taken as the second round of PCR template, and the second round of amplification was performed with the position-specific primers Pps-GGL / Pgs-GG2 and Pps-GG2 / Pgs-GGR, respectively, and the target band was detected by agarose gel electrophoresis. Then the 6a and 6b expression cassettes were recovered by PCR product purification kit, and 20 ng of each expression cassette was taken, and the expression cassette was connected to the pYLCRISPR / Cas9Pubi-H plasmid (donated by Professor Liu Yaoguang's team of South China Agricultural University) using Bsa I and T4 DNA ligase. The connection system is shown in the following table:

[0035]

[0036] After the connection system is configured, the enzyme cutting connection is performed by using temperature cycle, totally 15 cycles: 37℃, 5min; 10℃, 5min, 20℃, 5min; finally 37℃, 5min. After the connection is completed, half of the connection product is introduced into the E. coli DH5α competent cell by heat shock method, and after overnight culture, a single colony is picked and verified by PCR using primer pair SP1 / SP2. Three positive clones are selected for sequencing detection, and SP2 is used as the sequencing primer. After sequencing verification, the sequence of the expression cassette is correct, and the verified clone is the OsGLP3-7 gene editing vector.

[0037] The primer sequences used in the above steps are as follows:

[0038] OsGLP3-7-6aF: GTGCACTCCATTGATGATGACGGCAGCCAAGCCAGCA (SEQ ID NO. 5);

[0039] OsGLP3-7-6aR: TCATCATCAATGGAGTGCACGTTTTAGAGCTAGAAAT (SEQ ID NO. 6);

[0040] OsGLP3-7-6bF: GAACGGGTTGTCCGTGCTGCCAACACAAGCGGCAGC (SEQ ID NO. 7);

[0041] OsGLP3-7-6bR: CAGCACGGACAACCCGTTCGTTTTAGAGCTAGAAAT (SEQ ID NO. 8);

[0042] U-F: CTCCGTTTTACCTGTGGAATCG (SEQ ID NO. 9);

[0043] gR-R: CGGAGGAAAATTCCATCCAC (SEQ ID NO. 10);

[0044] Pps-GGL: TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG (SEQ ID NO. 11);

[0045] Pgs-GGR: AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC (SEQ ID NO. 12);

[0046] Pgs-GG2: AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC (SEQ ID NO. 13); Pps-GG2: TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG (SEQ ID NO. 14);

[0047] SP1: CCCGACATAGATGCAATAACTTC (SEQ ID NO. 15);

[0048] SP2: GCGCGGTGTCATCTATGTTACT (SEQ ID NO. 16).

[0049] 2. Obtaining of homozygous lines of OsGLP3-7 gene editing

[0050] The constructed OsGLP3-7 gene editing plasmid was selected and sent to Wuhan Boyuan Biotechnology Co., Ltd. for rice genetic transformation, and the transformation receptor was Nipponbare. A total of 20 T0 generation transgenic plants were obtained. The DNA of the transgenic plant leaves was extracted by the cetyltrimethylammonium bromide (CTAB) method, and then the genomic sequence on both sides of the editing site was amplified by using the OsGLP3-7JC-F / OsGLP3-7JC-R primer. The gene editing of OsGLP3-7 was detected by DNA sequencing of Shanghai Sunway Biotech Co., Ltd., and the homozygous mutant strain was selected for subculture.

[0051] The OsGLP3-7 gene homozygous mutant strain was subcultured, and the T1 generation strain was obtained by selfing. DNA was extracted, and the primer HygBioF and HygBioR were used for amplification. The amplification product was detected by agarose gel electrophoresis. The amplification band was clear for the offspring containing the hygromycin marker gene; the single plant without the hygromycin marker was screened, and the DNA of the hygromycin marker-free strain was used as a template to amplify the OsGLP3-7 gene fragment by using the primer pair OsGLP3-7JC-F / OsGLP3-7JC-R, and the OsGLP3-7 knockout mutant strain was screened by sequencing. A total of 2 base deletion homozygous lines Crispr-5 and Crispr-10 were obtained in this experiment. Figure 1), Crispr-5 deleted 238 bases (CACCGGAAACATGTCGGCGGCGCCATTGCTCGTCTTGACGGTGGCGGTGCTCGCCGT GCTAGCCTCCACCTGCGCCGCCGACCCAGAGCCGATACAAGACTTCTGCGTGGCCGTGCCCCGTGCCGGCGGCGAGGCGTCGCCGGCATACCCGGGCTTCCCGTGCAAGCCGGCGTCCGCGGTCGTCTCCGACGACTTCTTCTTCGCCGGGCTCGCCGCCGCCGGCAGCACGGACAACCCG), Crispr-10 deleted 1 base (G), all of which resulted in a frameshift mutation in the encoded amino acid, and the OsGLP3-7 gene was knocked out.

[0052] HygBioF: ACGGTGTCGTCCATCACAGTTTGCC (SEQ ID NO. 17);

[0053] HygBioR: TTCCGGAAGTGCTTGACATTGGGGA (SEQ ID NO. 18);

[0054] OsGLP3-7 JC-F: CGAGGGCCTGTCAATTTCT (SEQ ID NO. 19);

[0055] OsGLP3-7 JC-R: CGAGGGCCTGTCAATTTCT (SEQ ID NO. 20).

[0056] 3. Drought tolerance phenotype identification of OsGLP3-7 knockout plant seedlings

[0057] Three-leaf stage wild type Nipponbare and OsGLP3-7 knockout plant seedlings, which were cultivated in ordinary soil (from the rice field of the test base), were used for drought tolerance phenotype identification at the seedling stage. The three-leaf stage rice seedlings (without clear water on the surface of the soil) were placed in a small artificial climate chamber (28°C, 12h light / 12h darkness) for cultivation, and no water was poured during the cultivation to allow natural drought. After 10 days of water deprivation, the seedlings were re-watered and cultivated for another 6 days, and then the survival rate was counted. Compared with the wild type plants, the drought tolerance of the OsGLP3-7 knockout plants was significantly improved ( Figure 2 ) at the seedling stage.

[0058] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. The application of the rice OsGLP3-7 gene in regulating drought resistance during rice seedling stage, characterized by: The nucleotide sequence of the OsGLP3-7 gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.

2. Knocking out the OsGLP3-7 gene and disrupting its biological function improves the drought resistance of rice seedlings.

2. A molecular breeding method for improving drought resistance in rice seedlings, characterized in that, The OsGLP3-7 gene in rice was knocked out using the CRISPR / Cas9 editing system to disrupt its biological function, thereby obtaining a line with improved drought resistance in the seedling stage. The nucleotide sequence of the OsGLP3-7 gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.

2.

3. The molecular breeding method for improving drought resistance in rice seedlings according to claim 2, characterized in that, An OsGLP3-7 gene editing vector was constructed using CRISPR / Cas9 technology and transformed into rice to knock out the OsGLP3-7 gene in rice, thereby disrupting the biological function of the OsGLP3-7 gene. After screening of the progeny, rice lines with improved drought resistance in the seedling stage were obtained.

4. The molecular breeding method for improving drought resistance in rice seedlings according to claim 3, characterized in that, Includes the following steps: S1. Construction of editing vector: Based on CRISPR / Cas9 editing technology, two target sequences were selected in the OsGLP3-7 coding region. Then, two sgRNA expression cassettes containing the target sequences of the OsGLP3-7 coding region were cloned into the binary vector pYLCRISPR / Cas9Pubi-H, thereby forming a gene editing vector. S2. The editing vector was transferred into rice plants using Agrobacterium-mediated genetic transformation. S3. Based on the OsGLP3-7 gene sequence of the recipient variety, screen for homozygous mutants of the OsGLP3-7 gene, and then propagate these mutants to obtain lines with improved drought resistance in the seedling stage.

5. The molecular breeding method for improving drought resistance in rice seedlings according to claim 4, characterized in that, Two target sequences designed in the coding region of the OsGLP3-7 gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

6. The molecular breeding method for improving drought resistance in rice seedlings according to claim 4, characterized in that, Two sgRNA expression cassettes were constructed using primers OsGLP3-7-6aF / OsGLP3-7-6aR and OsGLP3-7-6bF / OsGLP3-7-6bR via a repeat stacking method. The sequences of OsGLP3-7-6aF, OsGLP3-7-6aR, OsGLP3-7-6bF, and OsGLP3-7-6bR are shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively.

7. The molecular breeding method for improving drought resistance in rice seedlings according to claim 4, characterized in that, The rice plants used were of the Nipponbare rice variety.

8. The molecular breeding method for improving drought resistance in rice seedlings according to claim 4, characterized in that, Mutants of the OsGLP3-7 gene were screened using primers OsGLP3-7JC-F / OsGLP3-7JC-R, where the sequence of OsGLP3-7JC-F is shown in SEQ ID NO.19 and the sequence of OsGLP3-7JC-R is shown in SEQ ID NO.20.

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