Application of Rice OsWAK3 Gene in Improving Salt Tolerance in Rice

Abstract

The present invention provides a method for identifying a rice OsWAK3 salt-tolerance gene and its application in improving the salt-tolerance trait of rice. The present invention identifies a new salt-tolerance gene OsWAK3, and after knocking out the WAK3 gene in the ZH11 variety, it is found that the knockout strain material becomes a salt-sensitive material compared with the ZH11 wild-type material, proving that the OsWAK3 gene is indeed a salt-tolerance gene, and providing a new resistance genetic resource for the breeding of rice salt-tolerance varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of rice molecular breeding and genetic engineering, and more specifically, relates to a rice OsWAK3 gene identification method and an application thereof in improving rice salt tolerance traits. Background Art

[0002] Rice (Oryza sativa L.) is the staple food of more than half of the world's population and one of my country's important food crops. The increasing degree of soil salinization has seriously restricted land use and the production and development of food crops. About 20% of the world's irrigated land is affected by salt stress. About 200 million mu of saline-alkali land in my country has the potential to grow rice. The development and utilization of saline-alkali land is of great strategic significance to ensure my country's food security. Rice is a moderately salt-sensitive crop and the preferred food crop for saline-alkali land improvement. Cultivating and promoting salt-alkali-tolerant rice varieties is the most economical and effective measure to expand the development and utilization of saline-alkali land and increase the rice planting area and yield. The identification of rice salt-tolerant genes and the study of molecular mechanisms, as well as the use of molecular design breeding methods to cultivate strong salt-alkali-tolerant rice varieties, are very meaningful to ensure the safe production and sustainable development of rice. Summary of the invention

[0003] The object of the present invention is to provide a method for identifying a rice OsWAK3 salt-tolerance gene and its application in improving the salt-tolerance trait of rice. The ID of the rice OsWAK3 gene is LOC_Os01g20880, its CDS nucleotide sequence is shown in sequence 1, and the encoded amino acid sequence is shown in sequence 2. After knocking out the WAK3 gene in the ZH11 variety, it was found that the knockout strain material became a salt-sensitive material compared with the ZH11 background material, proving that the OsWAK3 gene is indeed a salt-tolerance gene, providing a new resistance genetic resource for the breeding of rice salt-tolerant varieties. To achieve the above-mentioned invention purpose, the present invention adopts the following technical scheme as follows.

[0004] The first aspect of the present invention is the application of rice OsWAK3 in improving the salt tolerance of rice. The CDS nucleotide sequence of the rice OsWAK3 gene is shown in Sequence 1. The encoded amino acid sequence is shown in Sequence 2.

[0005] The second aspect of the present invention is to enhance the gene OsWAK3 in rice by genetic engineering means, so that the function of the gene OsWAK3 is enhanced or the expression level is increased.

[0006] In the third aspect of the present invention, a method for identifying the salt-tolerant gene of rice OsWAK3 is provided: an F2 genetic population is constructed by sea rice 86 and Dianjing You No. 1, and F2 individuals with extreme phenotypes (salt-sensitive and salt-tolerant) are selected in the F2 population to prepare a salt-tolerant and salt-sensitive mixed pool of BSA, and DNA sequencing is performed on the sea rice 86, Dianjing You No. 1 parents and the BSA extreme phenotype F2 mixed pool, a total of 4 samples. The four mixed pools are compared with the sea rice 86 reference genome. A total of 3,532,549 variant sites were identified, and based on these variant sites, 5 QTL sites were found on chr01, chr04 and chr06, respectively, which were named qST.1.1, qST.1.2, qST.1.3, qST.4.1, and qST.6.1. A WAK3 gene was found in the qST.1.2 interval of chromosome 1, and the gene was upregulated under salt stress conditions, which shows that the gene may be involved in salt stress response. Through comparison, it was found that the ID in the "Nipponbare" genome was LOC_Os01g20880.

[0007] A fourth aspect of the present invention provides a method for verifying the function of the rice OsWAK3 salt-tolerance gene, wherein the CDS nucleotide sequence of the OsWAK3 gene is shown in Sequence 1, and the method comprises the following steps:

[0008] (1) Using primers of sequence 3 and sequence 4, the OsWAK3 vector of CRISPR / Cas9 was constructed;

[0009] (2) The CRIPSR / Cas9-OsWAK3 vector was transferred into rice through callus transformation, and the material was gene-edited to obtain gene-edited rice oswak3 (23a, 27a, 29a);

[0010] (3) using the primers described in SEQ ID NO: 5 and SEQ ID NO: 6 to perform PCR amplification on the genomic DNA of the gene-edited rice oswak3;

[0011] (4) Sequencing the PCR products using sequence 5 to obtain the gene editing results and obtain knockout strains 23a, 27a, and 29a;

[0012] (5) Rice and T2 generation homozygous knockout lines 23a, 27a, and 29a were cultured in hydroponic nutrient solution for 14 days and then subjected to salt stress using 100 mmol / L NaCl. The results showed that the knockout lines 23a, 27a, and 29a showed a salt-sensitive phenotypic response.

[0013] Preferably, the rice is ZH11.

[0014] The functional verification results showed that knocking out the OsWAK3 gene in rice would turn the ZH11 knockout material into a sensitive material, thus proving that the OsWAK3 gene is a rice salt-tolerance gene and provides a new resistance genetic gene resource for rice salt-tolerance breeding.

[0015] Beneficial effects: The present invention is the first to discover the salt-tolerant gene OsWAK3 in rice. By knocking out the WAK3 gene in the Zhonghua 11 (ZH11) variety, it was found that the knockout strain material became a salt-sensitive material compared with the ZH11 background material, proving that the OsWAK3 gene is indeed a salt-tolerant gene, providing a new resistance genetic resource for the breeding of salt-tolerant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only illustrate the embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 QTL locus map for BSA analysis of Haidao 86 genetic population (the red line is p=1e -6 );

[0018] Figure 2 The LOC_Os01g20880 gene analysis and gene expression analysis in 277 rice populations;

[0019] Figure 3 The editing status of the gene-edited OsWAK3 rice material;

[0020] Figure 4 This is a diagram showing the phenotypic results of Zhonghua 11 (ZH11) and Oswak3 gene-edited materials (23a, 27a and 29a) after salt stress. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0024] Example 1 QTL identification for salt tolerance related traits

[0025] The salt-tolerant and salt-sensitive pools of BSA were prepared by constructing the F2 genetic population of Haidao 86 (from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences) and Dianjing You 1 (from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences), and selecting F2 individuals with extreme phenotypes (salt-sensitive and salt-tolerant) in the F2 population. DNA sequencing was performed on a total of four samples, including Haidao 86, Dianjing You 1, and the extreme phenotype F2 mixed pool of BSA (R01: Dianjing You 1; R02: Haidao 86; R03: Salt-tolerant pool; R04: Salt-sensive pool). The four mixed pools were aligned with the Haidao 86 reference genome. As expected, R02 had the highest alignment rate and R01 had a lower alignment rate (Table 1), further demonstrating the reliability of the experimental results.

[0026] Table 1 Alignment of the sequenced parents and mixed pools with the SR86 genome

[0027]

[0028] bcftools was further used to identify SNPs and InDels in the four sequencing pools (R01-R04). The identification results were filtered using the parameters %QUAL<10 || (RPB<0.1&&%QUAL<15) || (AC<2&&%QUAL<15) || MQ<30 || MQSB<=0.1. Finally, 3,532,549 variant sites were identified in the four sequencing pools, including 3,251,030 SNPs and 291,352 Indels, of which 163,380 variant sites were located in the exon region, and 7,637 variant sites caused large-effect mutations such as frameshift mutations, loss or gain of start or stop codons (Table 2).

[0029] Table 2 Variant site annotation

[0030]

[0031] The G' Value between R03 and R04 was calculated and visualized based on the variant sites. Five QTL loci were found on chr01, chr04 and chr06, respectively, and were named qST.1.1, qST.1.2, qST.1.3, qST.4.1, and qST.6.1 (Table 3, Figure 1 ).

[0032] Table 3 Identification of QTL loci for salt tolerance in Haidao 86

[0033]

[0034] Example 2 Prediction of candidate salt-tolerant genes

[0035] A WAK3 gene was found in the qST.1.2 interval of chromosome 1, and its expression was upregulated under salt stress conditions ( Figure 2 ). Combining the salt tolerance phenotypes, genotypes and variant site annotations of 277 Asian rice varieties, it was found that LOC_Os01g20880 among the differentially expressed genes was affected by a "high effect" mutation and was further identified as an important candidate salt tolerance gene. The LOC_Os01g20880 gene region has 24 SNP sites, including a mutation site that causes premature termination of the gene, 5 non-synonymous mutation sites, 4 mutation sites located in the 5' UTR, and other mutation sites are located in the intron region or are synonymous mutation sites. These 24 mutation sites divide the 277 rice materials into three haplotypes, among which the haplotype material with premature termination mutation sites has a significantly lower plant height under salt stress than other types of haplotype materials ( Figure 2 ). Based on this, it can be inferred that the termination mutation may have caused the loss of function of the gene, thereby leading to a decrease in rice salt tolerance.

[0036] Example 3 OsWAK3 gene function verification

[0037] 1. The CDS nucleotide sequence of the OsWAK3 gene of ZH11 is shown in SEQ ID NO:1.

[0038] 2. Use the two primers of sequence 3 and sequence 4 to construct the OsWAK3 vector of CRISPR / Cas9;

[0039] 3. The CRIPSR / Cas9-OsWAK3 vector was transferred into ZH11 rice through callus transformation, and the material was gene-edited to obtain gene-edited rice oswak3 (23a, 27a, 29a);

[0040] 4. Use primer sequences 5 and 6 to perform PCR amplification on the genomic DNA of gene-edited rice oswak3; the amplification procedure is as follows:

[0041]

[0042] 5. Then, the PCR products were sequenced using J1F to obtain the gene editing results, and the knockout strains 23a, 27a and 29a were obtained ( Figure 3 ).

[0043] 6. The ZH11 wild-type material and the T2 generation homozygous knockout strains 23a, 27a and 29a were cultured in hydroponic nutrient solution for 14 days, and then subjected to salt stress treatment with 100 mmol / L NaCl. The results showed that the knockout strains 23a, 27a and 29a showed salt-sensitive phenotypes ( Figure 4 ),from Figure 4 It can be seen that the leaves of knockout lines 23a, 27a and 29a wilt due to water loss compared to wild-type materials. This indicates that knocking out the OsWAK3 gene in ZH11 rice will turn the ZH11 knockout material into a sensitive material, thus proving that the OsWAK3 gene is a rice salt-tolerance gene, providing a new resistance genetic gene resource for rice salt-tolerance breeding.

[0044] CDS sequence of OsWAK3 (sequence 1)

[0045]

[0046] sequence2:

[0047] MESCVCACAWPTAPTVVALFATGYEEGVAEAPHDVDPMTNGHVVFQRVENNCSLRYFTENEIRQITRGYSILLGKGSFGKVYKGMLDGRCPVAVKRYIHGTRKEEFAKEVIVHSQINHKNVVRLLGCCTEENALMIVMEFICNGNLNDIL HCSNTNGRVPFSLGKRLDIAIEVAEVLWCMHSMYNPVLHGDIKPANILVDENLSPKLSDFGIARLLCANGAQHTNNIIGSIGYVDPAFCMNGILTPKSDVYSFGVVLLEIITRKKAVDGTITLAQRFTEAVEQGKKVMHLFDEDINNTKN

[0048] *

[0049] J228226-A1-F: 5'CCATCTCCGGCATCCTTCTC3' (sequence 3)

[0050] J228226-A1-R: 5'ACGTCCATTTGTGTTGCTGC3' (sequence 4)

[0051] J1F: CCATCTCCGGCATCCTTCTC (sequence 5)

[0052] J1R: ACGTCCATTTGTGTTGCTGC (sequence 6)

[0053] In summary, the present invention identified a new salt-tolerance gene OsWAK3 for the first time, and after knocking out the WAK3 gene in the ZH11 variety, it was found that the knockout strain material became a salt-sensitive material compared with the ZH11 background material, proving that the OsWAK3 gene is indeed a salt-tolerance gene, providing a new resistance genetic resource for the breeding of salt-tolerant rice varieties.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of rice OsWAK3 gene in regulating rice salt-sensitive traits, characterized in that: The rice OsWAK3 gene is knocked out to obtain salt-sensitive rice. The CDS nucleotide sequence of the rice OsWAK3 gene is shown in Sequence 1.

2. The use according to claim 1, characterized in that The amino acid sequence of the rice OsWAK3 gene is shown in Sequence 2.

3. The use according to claim 1, characterized in that The rice is ZH11.

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