Rice salt tolerance gene SAT14 and application thereof

Abstract

The application provides a rice salt tolerance gene SAT14 and application thereof, relates to the technical field of biology, and the transgenic plant of the SAT14 gene constructed by the application improves the salt tolerance of rice. Through functional interpretation of the SAT14 gene, the genetic mechanism of the salt tolerance of rice is further clarified, and a foundation is laid for breeding of a new salt-tolerant rice variety or variety improvement.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the rice salt tolerance gene SAT14 and its applications. Background Technology

[0002] Rice is a salt-sensitive plant, and ordinary rice cannot achieve high yields in saline-alkali land, which limits the further expansion of rice planting area in my country. Improving the salt tolerance of rice is of great significance for increasing the utilization rate of saline-alkali land, expanding limited arable land resources, increasing total grain production, and ensuring the absolute security of my country's food supply.

[0003] Therefore, identifying rice salt-tolerant genes and then using modern molecular breeding techniques to improve rice salt tolerance is a problem that urgently needs to be solved by those skilled in the art for expanding the planting area of ​​rice in saline-alkali land and achieving high and stable rice yields in saline-alkali land. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention proposes a rice salt tolerance gene SAT14 and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A rice salt tolerance gene, SAT14, having a nucleotide sequence as shown in SEQ ID NO.1.

[0007] The SAT14 gene may also have a sequence that encodes a protein with the same function by adding, substituting, or deleting one or more nucleotides in the nucleotide sequence defined in SEQ ID NO.1.

[0008] Another object of the present invention is to provide a rice salt-tolerant protein SAT14, wherein the SAT14 protein has the amino acid sequence shown in SEQ ID NO.2.

[0009] The SAT14 protein may also be a protein with one or more amino acids added, substituted, or deleted in the amino acid sequence defined by SEQ ID NO.2 and having the same function.

[0010] Another object of the present invention is to provide a biomaterial for reducing the expression level of the SAT14 gene or inhibiting the activity of the SAT14 protein, wherein the biomaterial is a recombinant vector capable of inhibiting the SAT14 gene or the SAT14 protein, or an expression cassette containing the recombinant vector, or a recombinant microorganism containing the expression cassette.

[0011] As a preferred embodiment, the expression cassette is SAT14-Cas9; the recombinant microorganism is an engineered bacterium or host cell used by those skilled in the art in the process of transgenic transformation.

[0012] Another objective of this invention is to provide the application of the aforementioned biological material in the cultivation of rice germplasm with enhanced salt tolerance, so as to serve as a screening marker in the rice breeding process for screening salt-tolerant germplasm resources.

[0013] Another objective of this invention is to provide a method for improving the salt tolerance of grass plants, comprising the following steps: reducing the expression level of the SAT14 gene or inhibiting the activity of the SAT14 protein through genetic engineering technology, and cultivating the transformed grass plant cells into plants.

[0014] As a preferred option, the grass plant is rice.

[0015] The technical effects of this invention are as follows: This invention provides a rice salt tolerance gene SAT14. Changes in the expression level of the SAT14 gene can cause changes in the salt tolerance of rice, providing a new gene resource for breeding salt-tolerant rice, and has great application prospects in rice breeding. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the plasmid of the pUbi-SAT14-Flag overexpression vector in an embodiment of the present invention;

[0017] Figure 2 This is a schematic diagram of the plasmid of the SAT14-Cas9 gene knockout vector in an embodiment of the present invention.

[0018] Figure 3 This is an identification diagram of the SAT14 transgenic plant in an embodiment of the present invention;

[0019] Figure 4 This is a schematic diagram illustrating the salt tolerance identification results of transgenic plants in an embodiment of the present invention. Detailed Implementation

[0020] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0021] Example:

[0022] 1. Construction of rice SAT14 gene overexpression vector

[0023] 1.1 Amplification of the Target Fragment: Based on the SAT14 gene sequence published on the rap-db website (https: / / rapdb.dna.affrc.go.jp / ), specific primers for amplifying the full-length SAT14 gene sequence were designed. The primer sequences are as follows:

[0024] OESAT14-F: 5'-caggagctcggtaccATGGAGGGTGGAGGCGCAAA

[0025] OESAT14-R: 5'-catggatccggtaccGGAAGCAGTCGGAATGGCAC

[0026] Using cDNA from wild-type Zhonghua 11 as a template, the full-length cDNA of the target gene was amplified.

[0027] 1.2 Overexpression Vector Construction: The overexpression vector pUbi-Flag was digested with KpnI, and the recovered product was ligated with recombinase and transformed into E. coli DH5α competent cells. After single colonies grew, sequencing was performed for verification. After successful sequencing, the pUbi-SAT14-Flag overexpression vector was obtained. A schematic diagram of its plasmid is shown below. Figure 1 As shown.

[0028] 2. Construction of rice SAT14 gene knockout vector

[0029] Based on the principles of the CRISPR-Cas9 system, the online gene knockout target site prediction and primer design website CRISPRdirect (https: / / crispr.dbcls.jp / ) was used to select target sites and design primers on the SAT14 gene. Following the methods provided by published CRISPR / Cas9 gene editing systems, a gene editing vector was constructed to obtain the SAT14-Cas9 gene knockout vector. A schematic diagram of its plasmid is shown below. Figure 2 As shown. For detailed steps, please refer to the reference (Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Liu Y. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant, 2015, 8: 1274-1284).

[0030] 3. Construction of transgenic plants

[0031] 3.1 Genetic transformation of rice callus: 2 μl of pUbi-SAT14-Flag and SAT14-Cas9 plasmids were added to Agrobacterium EHA105 competent cells, respectively. The cells were incubated on ice for 5 minutes, flash-frozen in liquid nitrogen for 5 minutes, incubated in water at 37°C for 5 minutes, and then on ice for 5 minutes. 750 μl of antibiotic-free LB broth was added to the competent cells, and the cells were incubated on a shaker at 28°C for 2 hours. 50 μl of the bacterial culture was then spread onto LB agar plates with the corresponding antibiotic resistance and incubated at 28°C for 2 days. Positive colonies were screened and identified.

[0032] 3.2. Transgenic plants of generation T0 were obtained by Agrobacterium-mediated transformation of callus tissue of Zhonghua 11, and the expression level of SAT14 gene was analyzed.

[0033] All the various culture media involved (induction medium, screening medium, differentiation medium, LB medium) are conventional culture media.

[0034] The results showed that, compared with the wild type, the overexpressing transgenic plants OESAT14 The expression level of the SAT14 gene was significantly increased; knockout transgenic plants CrSAT14 The expression level of the SAT14 gene is reduced, as shown in the identification diagram. Figure 3 As shown.

[0035] 4. Evaluation of salt tolerance of overexpression and knockout lines

[0036] Wild-type Zhonghua 11 and transgenic materials were soaked and germinated, then sown and hydroponically cultured in nutrient solution for 14 days. Rice plants were then treated with a nutrient solution containing 120 mM NaCl for 7 days, followed by 7 days of culture in normal nutrient solution. Growth of each line was then recorded. The study found that under salt stress: wild-type Zhonghua 11 plants reached the 2-3 leaf stage, while SAT14-overexpressing lines withered and died at the 2-leaf stage; SAT14 knockout lines reached the 4-5 leaf stage. The identification results are as follows: Figure 4 As shown, SAT14 negatively regulates rice salt tolerance.

[0037] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent structures made using the content of the present invention specification, whether directly or indirectly applied to other related technical fields, are similarly within the patent protection scope of the present invention.

Claims

1. Use of a biological material in breeding of a rice germplasm with improved salt tolerance, characterized in that, The biomaterial is a recombinant vector capable of reducing the expression level of the SAT14 gene or inhibiting the activity of the SAT14 protein; the nucleotide sequence of the SAT14 gene is shown in SEQ ID NO.1, and the amino acid sequence of the SAT14 protein is shown in SEQ ID NO.

2.

2. A method for improving salt tolerance in a plant of the family Gramineae, characterized by, The method includes the following steps: reducing the expression level of the SAT14 gene or inhibiting the activity of the SAT14 protein through genetic engineering technology, and cultivating the transformed gramineous plant cells into plants; the nucleotide sequence of the SAT14 gene is shown in SEQ ID NO.1, the amino acid sequence of the SAT14 protein is shown in SEQ ID NO.2, and the gramineous plant is rice.

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