Application of Arabidopsis thaliana AtFLZ13 Gene in Plant Salt-Resistant Breeding

Abstract

The present invention discloses the application of Arabidopsis thaliana AtFLZ13 gene in regulating rice resistance to high salt and / or cultivating rice varieties resistant to high salt stress, belonging to the technical field of genetic engineering. The nucleotide sequence of the AtFLZ13 gene encodes an amino acid sequence as shown in SEQ ID NO.2. By overexpressing AtFLZ13, the present invention can improve the resistance of rice to high salt stress, and can be applied to plant genetic engineering breeding, providing a new method for the creation or improvement of new rice germplasms resistant to high salt.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to application of Arabidopsis thaliana AtFLZ13 gene in plant salt stress resistance breeding. Background Art

[0002] Globally, nearly 932 million hectares of land are affected by salinization, leading to land degradation, reduced agricultural productivity, and even food security concerns. China has approximately 36 million hectares of saline-alkali land, accounting for approximately 5% of the country's usable land area. Most of this is saline-alkali wasteland, with only about one-fifth being arable land. This leaves 17.5 million hectares of land under potential salinization threat. Therefore, soil salinization has become a major limiting factor for crop growth and production in my country and globally. Rice, one of my country's most important food crops, is severely inhibited by high salt stress, which can lead to yield reductions. Therefore, identifying plant salt-tolerance genes and using them to breed new salt-tolerant plant varieties is of great significance for ensuring global food security, promoting sustainable agricultural development, achieving sustainable land resource utilization, and improving the ecological environment. Summary of the Invention

[0003] The purpose of the present invention is to provide an application of an Arabidopsis thaliana AtFLZ13 gene in regulating plant resistance to salt stress and / or cultivating salt stress-resistant plant varieties.

[0004] This invention demonstrates the regulatory effect of the Arabidopsis thaliana AtFLZ13 gene on salt stress resistance in Arabidopsis thaliana and rice through transgenic experiments and phenotypic analysis. The AtFLZ13 gene encodes the nucleotide sequence represented by the amino acid sequence shown in SEQ ID NO. 2. This gene is expected to be applied in genetic engineering plant breeding, providing a theoretical basis for the creation or improvement of new plant germplasm with salt stress tolerance. Given the degeneracy of codons, modifications to the bases in the nucleotide sequence without altering the amino acid sequence are also within the scope of this invention.

[0005] In one aspect, the present invention provides an application of an Arabidopsis thaliana AtFLZ13 gene in regulating plant resistance to salt stress. The AtFLZ13 gene encodes a nucleotide sequence having an amino acid sequence as shown in SEQ ID NO.2.

[0006] Furthermore, the regulation of plant resistance to salt stress is to improve the plant resistance to salt stress by overexpressing the AtFLZ13 gene.

[0007] In a preferred embodiment, the present invention improves the resistance of monocotyledonous plants to salt stress by overexpressing the AtFLZ13 gene.

[0008] Furthermore, the present invention improves the resistance of grass plants to salt stress by overexpressing the AtFLZ13 gene.

[0009] Furthermore, the present invention improves the resistance of Oryza subfamily plants to salt stress by overexpressing the AtFLZ13 gene.

[0010] Furthermore, the present invention improves the resistance of rice plants to salt stress by overexpressing the AtFLZ13 gene.

[0011] Furthermore, the present invention improves the resistance of rice to salt stress by overexpressing the AtFLZ13 gene.

[0012] In another preferred embodiment, the present invention improves the resistance of dicotyledonous plants to salt stress by overexpressing the AtFLZ13 gene.

[0013] Furthermore, the present invention improves the resistance of cruciferous plants to salt stress by overexpressing the AtFLZ13 gene.

[0014] On the other hand, the present invention provides a use of a protein encoded by the Arabidopsis thaliana AtFLZ13 gene in regulating plant resistance to salt stress, wherein the amino acid sequence of the AtFLZ13 protein is shown as SEQ ID NO.2.

[0015] Furthermore, the regulation of plant resistance to salt stress is to improve the plant resistance to salt stress by overexpressing the protein encoded by the AtFLZ13 gene.

[0016] In a preferred embodiment, the present invention improves the resistance of monocotyledonous plants to salt stress by overexpressing AtFLZ13 protein.

[0017] Furthermore, the present invention improves the resistance of grass plants to salt stress by overexpressing AtFLZ13 protein.

[0018] Furthermore, the present invention improves the resistance of Oryza subfamily plants to salt stress by overexpressing AtFLZ13 protein.

[0019] Furthermore, the present invention improves the resistance of rice plants to salt stress by overexpressing AtFLZ13 protein.

[0020] Furthermore, the present invention improves the resistance of rice to salt stress by overexpressing AtFLZ13 protein.

[0021] In another preferred embodiment, the present invention improves the resistance of dicotyledonous plants to salt stress by overexpressing AtFLZ13 protein.

[0022] Furthermore, the present invention improves the resistance of cruciferous plants to salt stress by overexpressing AtFLZ13 protein.

[0023] On the other hand, the present invention provides an application of the Arabidopsis thaliana AtFLZ13 gene in cultivating salt stress-resistant plant varieties, wherein the AtFLZ13 gene sequence is a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1, or a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0024] Furthermore, the breeding of salt stress resistant plant varieties is breeding of salt stress resistant plant varieties by overexpressing the AtFLZ13 gene.

[0025] In a preferred embodiment, the present invention cultivates salt stress-resistant monocotyledonous plant varieties by overexpressing the AtFLZ13 gene.

[0026] Furthermore, the present invention cultivates salt stress-resistant Gramineae plant varieties by overexpressing the AtFLZ13 gene.

[0027] Furthermore, the present invention cultivates salt stress-resistant Oryza subfamily plant varieties by overexpressing the AtFLZ13 gene.

[0028] Furthermore, the present invention cultivates salt stress-resistant rice varieties by overexpressing the AtFLZ13 gene.

[0029] Furthermore, the present invention cultivates salt stress-resistant rice varieties by overexpressing the AtFLZ13 gene.

[0030] In another preferred embodiment, the present invention cultivates dicotyledonous plant varieties resistant to salt stress by overexpressing the AtFLZ13 gene.

[0031] Furthermore, the present invention cultivates salt stress-resistant cruciferous plants by overexpressing the AtFLZ13 gene.

[0032] On the other hand, the present invention provides a use of a protein encoded by the Arabidopsis thaliana AtFLZ13 gene in cultivating salt stress-resistant plant varieties, wherein the AtFLZ13 gene encodes a nucleotide sequence having an amino acid sequence as shown in SEQ ID NO.2.

[0033] Furthermore, the method of cultivating salt stress-resistant plant varieties is to cultivate salt stress-resistant plant varieties by overexpressing AtFLZ13 protein.

[0034] In a preferred embodiment, the present invention cultivates salt stress-resistant monocotyledonous plant varieties by overexpressing AtFLZ13 protein.

[0035] Furthermore, the present invention cultivates salt stress-resistant Gramineae plant varieties by overexpressing AtFLZ13 protein.

[0036] Furthermore, the present invention cultivates salt stress-resistant Oryza subfamily plant varieties by overexpressing AtFLZ13 protein.

[0037] Furthermore, the present invention cultivates salt stress-resistant rice varieties by overexpressing AtFLZ13 protein.

[0038] Furthermore, the present invention cultivates salt stress-resistant rice varieties by overexpressing AtFLZ13 protein.

[0039] In another preferred embodiment, the present invention cultivates dicotyledonous plant varieties resistant to salt stress by overexpressing AtFLZ13 protein.

[0040] Furthermore, the present invention cultivates salt stress-resistant cruciferous plant varieties by overexpressing AtFLZ13 protein.

[0041] Compared with the prior art, the present invention has the following advantages:

[0042] 1) The present invention provides a novel method for regulating plant salt stress resistance and / or cultivating salt stress-resistant plant varieties, namely, regulating plant resistance to salt stress and / or obtaining salt stress-resistant plant varieties by overexpressing the AtFLZ13 gene or the protein encoded by it.

[0043] 2) The novel method for regulating plant resistance to salt stress and / or cultivating salt stress-resistant plant varieties provided by the present invention can be used for plants with different genetic backgrounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The method for regulating plant resistance to salt stress and / or cultivating salt stress-resistant plant varieties and its beneficial effects of the present invention are described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Figure 1 Shown is a schematic diagram of the expression cassette of the AtFLZ13 overexpression vector;

[0046] Figure 2 Shown is the expression analysis of the AtFLZ13 gene in transgenic rice; Nip represents Nipponbare; OE#3 and OE#6 represent overexpression plants.

[0047] Figure 3 Shown are the phenotypes of AtFLZ13 transgenic and Nip plants under salt stress treatment; Nip represents Nipponbare; OE#3 and OE#6 represent overexpression plants; Figure 3 A is the growth status of rice before and after treatment, Figure 3 B is the statistical results of survival rate. DETAILED DESCRIPTION

[0048] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0049] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0050] The nucleotide sequence of the Arabidopsis thaliana AtFLZ13 gene in the embodiment is shown in SEQ ID NO.1:

[0051] .

[0052] The amino acid sequence of Arabidopsis thaliana AtFLZ13 protein is shown in SEQ ID NO.2:

[0053] MILSKRPHLMIRKLSEMLVPRSRSAAIKPEEYTASPRSPLDLNFPSPVHSKRFGSGGVGLGIVAALEETSNGINRHDPVRYSGRFRCPEIDLSDEEYTYVTSPNGPTKVYY NDDGFELSENDYRRVHKPMVTVDEPPVIERQSVRGPTEFLSSCCLCKKKLQGKDIYMYKGEMGFCSAECRSVQIMNDERQEQCKTQVSRNADVLSSPYAAGQRLSAGVFVF.

[0054] Example 1 Construction of Arabidopsis thaliana AtFLZ13 gene overexpression vector

[0055] 0.5 g of Arabidopsis seedlings germinated and grown for 7 days on 1 / 2 MS solid medium were taken and total RNA was extracted using a plant RNA extraction kit (Magen). The target gene was then amplified. The specific experiment is as follows:

[0056] 1 μg of high-quality RNA (OD260 / OD280: 1.8-2.0; OD260 / OD230 ≈ 2.0) was reverse transcribed using a Novozymes reverse transcription kit to generate first-strand cDNA. PCR amplification was performed using the cDNA as a template using KOD FX high-fidelity enzyme (ToYoBo). The reaction system consisted of 10 μL of 2× PCR buffer, 2 μL of 2 mM dNTPs, 0.5 μL of F primer (5'-CTGATTAACAGGGATCCCCCATGATACTAAGCAAGAGACCTC-3'), 0.5 μL of R primer (5'-TCGAGACTAGTGGTACCCCCAAATACGAATACTCCGGCAGAT-3'), 1 μL of cDNA template, and 0.4 μL of KOD FX (1 U / μL). The volume was made up to 20 μL with water. The reaction conditions were: 98°C for 5 min, 35 cycles of 98°C for 15 sec, 56°C for 30 sec, and 68°C for 30 sec, and 68°C for 5 min. After the reaction, the PCR product was recovered using a gel recovery kit (Magen).

[0057] The pCAMBIA1300-GFP vector (modified from the pCAMBIA1300 vector and containing the Arabidopsis thaliana Ubiquitin 10 promoter and a GFP tag) was linearized with the restriction endonuclease SmaI (NEB). The digestion system consisted of 2 μg of vector, 2 μL of SmaI endonuclease, 10 μL of 10× rCutSmart buffer, and water to 100 μL. The digestion conditions were 25°C for 2 hours. The vector and PCR products were purified and recovered, and DNA concentrations were determined using a NanoDrop 2000. Recombination was performed using a recombination kit (Novozymes). The recombination system consisted of 14 ng of target fragment, 130 ng of vector fragment, 2 μL of 5× buffer, 1 μL of Exnase II, and water to 10 μL. The recombination conditions were 37°C for 30 minutes. The entire product was added to 100 μL of competent E. coli DH5α. The transformed product was then plated onto LB solid medium (containing kanamycin resistance at a concentration of 50 mg / L). The culture was incubated overnight at 37°C, and four single colonies were identified by colony PCR. Two positive clones were selected for sequencing, and positive clones containing the AtFLZ13 gene sequence were obtained. Finally, an overexpression vector containing the AtFLZ13 target gene was obtained ( Figure 1 ).

[0058] Example 2 Acquisition and identification of AtFLZ13 transgenic rice

[0059] The overexpression vector constructed in Example 1 was transformed into the japonica rice variety Nipponbare using Agrobacterium tumefaciens EHA105-mediated genetic transformation. T0-generation transgenic seedlings were obtained through selective culture, differentiation, rooting, and hardening. Reference: "Study on a High-Efficiency Agrobacterium-Mediated Rice Transformation System" by Zheng Jie; Hunan Agricultural Sciences, 2008, Vol. 2.

[0060] All transgenic seedlings were identified by PCR to amplify the GFP vector fragment. Ten positive transgenic seedlings were propagated to generate T1 and T2 generations. T2 seeds were germinated using a medium containing hygromycin. If all seeds grew normally, the strain was considered homozygous. Two transgenic lines (OE#3 and OE#6) were selected for qRT-PCR analysis and subsequently tested and analyzed.

[0061] The qRT-PCR identification process of the overexpression effect of AtFLZ13 gene in transgenic rice is as follows:

[0062] 1. Total RNA was extracted from rice leaves at the four-leaf stage using a plant RNA extraction kit (Magen). 1 μg of high-quality RNA (OD260 / OD280: 1.8-2.0; OD260 / OD230 ≈ 2.0) was reverse transcribed using a Novozymes reverse transcription kit to obtain first-strand cDNA.

[0063] 2. Using the cDNA from step 1 above as a template, the AtFLZ13 gene expression was detected using the primer pair AtFLZ13-qF (ATGGCAGCTGAATCCTCCCT) and AtFLZ13-qR (GCATCCAGCTCAATCAAACA). The rice housekeeping gene EF1α was detected using the primer pair OsEF1α-qF (TTTCACTCTTGGTGTGAAGCAGAT) and OsEF1α-qR (GACTTCCTTCACGATTTCATCGTAA) as an internal control. SYBR® Premix Ex Taq™ (TAKARA) was used as the quantitative PCR reagent and the CFX 96 (Bio-RAD) instrument was used. The reaction system consisted of 5 μL of 2× PCR buffer, 0.4 μL of qF primer, 0.4 μL of qR primer, 1 μL of cDNA template, and 3.2 μL of sterile water for a total reaction volume of 10 μL. Reaction program: 95°C 30 sec; 95°C 5 sec, 68°C 30 sec, 45 cycles.

[0064] The results are as follows Figure 2 As shown, the expression levels of the AtFLZ13 gene in the two selected strains were greatly increased, and these two overexpression strains were subsequently selected for experiments.

[0065] Example 3 Analysis of the Salt Stress Resistance Phenotype of AtFLZ13 Overexpressing Rice

[0066] Seeds of the homozygous overexpressing plants obtained in Example 2 and wild-type Nipponbare were broken from dormancy. After germination for two days, seedlings of uniform size were selected and sown in 96-well culture dishes. The seeds were then placed in a plant incubator (28°C, 12 h light / 12 h dark) for continued growth. After two weeks of growth, the seedlings were transferred to a solution containing 150 mM NaCl and cultured for eight days, followed by five days of recovery under normal conditions. Photos were taken and the survival rate was calculated. Three biological replicates were performed for each material.

[0067] Result analysis: The results before and after treatment are shown in Figure 3 Middle A, after treatment with 150 mM NaCl, the growth status of the overexpression plants was significantly better than that of the control group Nipponbare.

[0068] The survival rate results are shown in Figure 3 In B, after treatment with 150 mM NaCl, the average survival rates were: Nip: 29.2%; OE#3: 47.2%; OE #6: 68.0%.

[0069] >SEQ ID NO.1

[0070] ATGATACTAAGCAAGAGACCTCATCTAATGATCCGTAAATTGTCTGAGATGTTGGTTCCAAGAAGCAGATCTGCTGCTATTAAACCGGAGGAGTACACGGCCAGTCCGAGAAGTCCGTTGGATTTGAACTTCCCCTCCCCGGTTCACTCGAAGCGGTTCGGTTCTGGTGGTGTCGGTTTGGGTATTGTTGCTGCGTTGGAGGAAACTAGCAACGGGATTAACCGGCATGATCCGGTTCGTTACTCCGGGAGATTCAGGTGCCCGGAGATTGATCTGTCGGATGAGGAATACACTTATGTTACGAGCCCAAACGGGCCGACCAAAGTGTATTACAACGACGACGGGTTTGAATTGTCTGAAAATGATTATCGGAGAGTTCATAAACCGATGGTTACCGTCGATGAACCACCGGTTATTGAAAGACAGAGTGTTAGGGGTCCAACAGAGTTTCTGAGTTCGTGTTGCTTGTGTAAGAAGAAACTTCAAGGCAAAGACATATACATGTACAAAGGAGAGATGGGATTTTGTAGTGCCGAATGCAGATCAGTGCAAATAATGAATGACGAACGACAAGAGCAATGTAAAACGCAAGTTTCAAGAAACGCCGACGTTTTGAGCTCTCCTTACGCCGCCGGACAGAGATTATCTGCCGGAGTATTCGTATTTTAG。

[0071] >SEQ ID NO.2

[0072] MILSKRPHLMIRKLSEMLVPRSRSAAIKPEEYTASPRSPLDLNFPSPVHSKRFGSGGVGLGIVAALEETSNGINRHDPVRYSGRFRCPEIDLSDEEYTYVTSPNGPTKVYY NDDGFELSENDYRRVHKPMVTVDEPPVIERQSVRGPTEFLSSCCLCKKKLQGKDIYMYKGEMGFCSAECRSVQIMNDERQEQCKTQVSRNADVLSSPYAAGQRLSAGVFVF.

[0073] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to the above embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest possible manner consistent with the principles and novel features disclosed herein.

Claims

1. Application of Arabidopsis thaliana AtFLZ13 gene in regulating plant resistance to salt stress, characterized in that, The AtFLZ13 gene encodes an amino acid sequence shown in SEQ ID NO.2; the plant is rice; the regulation of the plant's resistance to salt stress is to improve the plant's resistance to salt stress by overexpressing the AtFLZ13 gene.

2. Use of the protein encoded by the Arabidopsis thaliana AtFLZ13 gene in regulating plant resistance to salt stress, characterized in that, The AtFLZ13 gene encodes an amino acid sequence shown in SEQ ID NO.2; the plant is rice; the regulation of the plant's resistance to salt stress is to improve the plant's resistance to salt stress by overexpressing the protein encoded by the AtFLZ13 gene.

3. Use of Arabidopsis thaliana AtFLZ13 gene in cultivating salt stress-resistant plant varieties, characterized in that, The AtFLZ13 gene encodes an amino acid sequence shown in SEQ ID NO.2; the plant is rice; the cultivation of a plant variety resistant to salt stress is to cultivate a plant variety resistant to salt stress by overexpressing the AtFLZ13 gene.

4. Use of the protein encoded by the Arabidopsis thaliana AtFLZ13 gene in cultivating plant varieties resistant to salt stress, characterized in that, The AtFLZ13 gene encodes an amino acid sequence shown in SEQ ID NO.2; the plant is rice; the cultivation of a plant variety resistant to salt stress is to cultivate a plant variety resistant to salt stress by overexpressing the protein encoded by the AtFLZ13 gene.

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