Drought-resistant gene bnaalaat1 of brassica napus and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and discloses a drought-resistant gene of Brassica napus BnaALaAT1 and application, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2, and the drought-resistant ability of the plant can be significantly improved after the gene is introduced into Arabidopsis thaliana.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the rapeseed drought-resistant gene BnaALaAT1 and its application. Background Technology

[0002] Drought stress has long been considered one of the most significant problems facing agricultural production worldwide, and it is one of the most prevalent and prominent issues inhibiting agricultural output. The damage of drought to crop yields exceeds the sum of other environmental stressors; even short-term drought stress can cause yield losses. In recent years, global greenhouse effect has exacerbated drought in some regions. Statistics show that more than one-third of the world's land area is semi-arid or arid. Even in semi-humid or even humid regions with sufficient water, droughts frequently occur for various reasons, whether temporary, seasonal, or periodic.

[0003] Traditional drought-resistant breeding of crops is hampered by long cycles, slow results, and a limited pool of drought-resistant germplasm resources, resulting in slow progress. The rapidly developing biotechnology breeding, especially transgenic technology, has broken down species barriers and provided new avenues for efficient drought-resistant breeding; however, obtaining drought-resistant gene resources is crucial for developing new drought-resistant transgenic crop varieties. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the gene BnaALaAT1 in improving plant drought resistance, so as to solve the current situation of drought resistance gene scarcity.

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

[0006] Primers were designed based on NCBI XM_013827516 (PREDICTED: Brassica napus alanineaminotransferase 1, mitochondrial) to clone the gene BnaALaAT1 from the rapeseed variety “Zhongshuang 11” (ZS11). The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0007] Application of gene BnaALaAT1 in improving plant drought resistance: In a specific embodiment of the present invention, gene BnaALaAT1 was transferred into Arabidopsis thaliana to obtain pure line plants of Arabidopsis thaliana with gene BnaALaAT1. After drought resistance identification, compared with wild-type Arabidopsis thaliana, Arabidopsis thaliana with gene BnaALaAT1 had robust root system, good growth and good drought resistance.

[0008] A method to improve plant drought resistance: construct an expression vector containing the gene BnaALaAT1, introduce the expression vector into Agrobacterium, transform plants with Agrobacterium to obtain T0 generation containing the gene BnaALaAT1, and obtain pure lines transfected with BnaALaAT1 through three consecutive generations of screening.

[0009] The beneficial effect of this invention lies in cloning a new drought-resistant gene from rapeseed, which was further transformed into Arabidopsis thaliana for drought resistance identification. The results showed that the Arabidopsis thaliana lines transformed with the BnaALaAT1 gene exhibited excellent drought resistance. Furthermore, BnaALaAT1 has a molecular weight of 1877 bp and encodes a protein containing 542 amino acids, facilitating genetic transformation. The acquisition of the drought-resistant gene in this invention provides a valuable genetic resource for the breeding of drought-resistant crops and new plant varieties (lines). Attached Figure Description

[0010] Figure 1 Electrophoresis diagram of the amplified products of the BnaALaAT1 gene. M: 2K Plus DNA Ladder; 1-2 are PCR products of the BnaALaAT1 gene.

[0011] Figure 2 The results of PCR identification of Agrobacterium with PS2300-BnaALaAT1 plasmid are shown. M: 2K Plus DNA Ladder, 1-2 are the bacterial culture numbers of single clones in culture.

[0012] Figure 3 The results show the molecular detection of positive Arabidopsis thaliana T3 generation plants transgenic with the BnaALaAT1 gene. M: 2K Plus DNA Ladder, 1-9 are PCR products of positive seedlings, "WT": wild-type Arabidopsis thaliana; "+": plasmid; "-": blank control.

[0013] Figure 4 The growth status of Arabidopsis thaliana T3 generation lines transformed with the BnaALaAT1 gene under artificially simulated drought stress. Detailed Implementation

[0014] The principles and features of the present invention are described below with reference to embodiments. The examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0015] Example 1: Cloning of the drought-resistant gene BnaALaAT1 in rapeseed

[0016] Using the rapeseed variety "Zhongshuang 11" (ZS11) as material, total RNA was extracted using a plant total RNA mini-extraction kit (Magen), and the first strand of cDNA was synthesized by reverse transcription using a cDNA kit. The gene fragment was then amplified using a PCR reaction system of I-5. TM 10 μl of 2×High-Fidelity Master Mix, 0.5 μl of 10 μM upstream primer (5'–ggatccATGCGGAGATTCGTTATTGGC–3'), 0.5 μl of 10 μM downstream primer (5'–gagctcCGGCATAGGTCTCTGTTGCT–3'), 1 μl of cDNA, 8 μl of ddH2O, for a total volume of 20 μl;

[0017] The amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 10 min. The PCR products were recovered using a gel extraction kit, and then ligated into the pTOPO vector to obtain the recombinant plasmid pTOPO-BnaALaAT1. This plasmid was transformed into competent *E. coli* cells, and colonies were selected for PCR amplification. The target band was detected by agarose gel electrophoresis (results are shown in the figure). Figure 1 After sequencing, the nucleotide sequence of the BnaALaAT1 gene is shown in SEQ ID NO.1, with a molecular weight of 1877 bp, and the amino acid sequence it encodes is shown in SEQ ID NO.2.

[0018] Example 2: Method for constructing the expression vector of the rapeseed drought resistance gene BnaALaAT1

[0019] DNA was extracted from the pTOPO-BnaALaAT1 recombinant plasmid containing the BnaALaAT1 gene and the plant expression vector PS2300 plasmid. The extracted plasmid was double-digested with BamHI and SacI restriction enzymes. The digestion system consisted of 1.5 μl BamHI, 1.5 μl SacI, 5 μl 10× buffer, 12 μl plasmid, and 30 μl ddH2O, for a total volume of 50 μl. The digestion was carried out at 37℃ for 5.5 h, and the target fragment was recovered and purified.

[0020] Next, the purified and recovered vector and target fragment were ligated using T4 ligase. The ligation system consisted of 0.5 μl of vector PS2300, 3.5 μl of target fragment, 0.5 μl of T4 ligase, and 0.5 μl of T4 buffer, for a total volume of 5 μl. Ligation was carried out at room temperature for 3 h. The resulting recombinant plasmid PS2300-BnaALaAT1 was digested with restriction endonucleases BamHI and SacI at 37 °C for 5.5 h and then detected by electrophoresis. The ligated recombinant plasmid PS2300-BnaALaAT1 was transformed into E. coli DH5α competent cells, colonies were selected for PCR amplification, and the target band was detected by agarose gel electrophoresis before expansion culture.

[0021] Example 3: Agrobacterium-mediated transformation and culture method in Arabidopsis thaliana

[0022] The recombinant plasmid PS2300-BnaALaAT1 was transformed into competent Agrobacterium GV3101 (Beijing TransGen Biotech Co., Ltd.) (identification results are shown in [link to documentation]). Figure 2 Agrobacterium tumefaciens was prepared, and Arabidopsis inflorescences were inoculated using the flower-dipping method. The inoculated plants were then covered with plastic film and cultured in the dark for 24 hours before being transferred to a greenhouse for conventional cultivation. Once mature, seeds were harvested from individual plants. The harvested Arabidopsis seeds were sterilized at 4°C and then sown in 1 / 2 MS solid medium containing the antibiotic Kan (50 μg / ml) to screen for positive seedlings. These positive seedlings were then transplanted into a culture medium (vermiculite: nutrient soil in a 1:1 volume ratio). After the seedlings matured, DNA was extracted from plant leaves and verified by PCR using the upstream and downstream primers for the BnaALaAT1 gene from Example 1. Screening with the antibiotic Kan was repeated, and PCR detection of the target gene was performed (see Example 1). Figure 3 ), until T3 generation Arabidopsis thaliana transgenic with BnaALaAT1 gene pure line was obtained.

[0023] Example 4: Identification of drought resistance of transgenic BnaALaAT1 gene

[0024] The T3 generation transgenic Arabidopsis thaliana plants obtained in Example 4 and wild-type Arabidopsis thaliana (Col-0) seeds were sown in 1 / 2 MS solid medium. After one week, they were transferred to 1 / 2 MS + 4% PEG solid medium and cultured in a light incubator for one week. The results showed that the transgenic Arabidopsis thaliana in 1 / 2 MS + 4% PEG solid medium grew significantly better than wild-type Arabidopsis thaliana, and its root system was also more robust (see...). Figure 4Wild-type Arabidopsis thaliana exhibited inhibited growth, with leaves drying, yellowing, and wilting, when grown in 1 / 2 MS + 4% PEG solid medium. In contrast, Arabidopsis thaliana transgenic with the BnaALaAT1 gene showed good drought resistance, surviving and growing well in the same medium. Therefore, the BnaALaAT1 gene can significantly improve drought resistance in Arabidopsis thaliana, and this gene could be used for drought-resistant breeding in plants or crops.

[0025] The above embodiments are the best implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Genes BnaALaAT1 The application of its encoded proteins in improving drought resistance in Arabidopsis thaliana, characterized in that, The gene BnaALaAT1 The nucleotide sequence of the protein is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. A method for improving the drought resistance of Arabidopsis thaliana, characterized in that, Constructing a gene BnaALaAT1 The expression vector was introduced into Agrobacterium, and Arabidopsis thaliana was transformed by Agrobacterium to obtain a gene-containing strain. BnaALAAT1 The T0 generation, obtained through three consecutive generations of screening, is a transitional generation. BnaALaAT1 The pure line, the gene BnaALaAT1 The nucleotide sequence is shown in SEQ ID NO.1.

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