Peanut bacterial wilt resistant NBS-LRR coding gene AhRRS3 and application thereof

Abstract

The invention provides a peanut bacterial wilt resistance NBS-LRR coding gene AhRRS3 and application thereof. The nucleotide sequence of the gene AhRRS3 is shown as SEQ ID NO.1, and the amino acid sequence of the protein coded by the gene AhRRS3 is shown as SEQ ID NO.2. The invention further provides a preparation method of the peanut bacterial wilt resistance NBS-LRR coding gene AhRRS3. According to the invention, an overexpression vector of AhRRS3 is constructed, arabidopsis thaliana is transformed, and the disease-resistant function of the arabidopsis thaliana is observed. Research results show that after being infected by ralstonia solanacearum, the disease-resistant variety of arabidopsis thaliana and a disease-susceptible strain of the overexpressed gene AhRRS3 show obvious disease resistance, the disease-susceptible strain is in a whole-plant withering and death state, and the results show that the overexpressed gene AhRRS3 can improve the bacterial wilt resistance of the plant. The invention provides high-quality gene resources and theoretical basis support for genetic engineering breeding of plant bacterial wilt resistance.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a peanut NBS-LRR gene encoding resistance to bacterial wilt. AhRRS3 And its applications. Background Technology

[0002] peanut( Arachis hypogaea Peanuts (Ralstonia solanacearum) are an important oilseed crop worldwide, rich in nutrients and an indispensable source of edible vegetable oil. The development of the peanut industry is of great significance to ensuring people's livelihood and food security. Bacterial wilt of peanuts is a major disease problem, caused by the soil-borne fungus Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum This fungus, *Ralstonia solanacearum*, infects peanut plants through the roots, causing wilting and death, severely limiting peanut yield. It also widely infects other important crops, such as tobacco, potatoes, peppers, and eggplants.

[0003] However, since there are currently no effective agents to eradicate bacterial wilt, the location and functional identification of bacterial wilt resistance genes in peanuts are crucial for identifying high-quality resistance gene resources and promoting the breeding of resistant varieties. This provides important application value for ensuring global grain and oil resources. Summary of the Invention

[0004] The purpose of this invention is to provide a peanut NBS-LRR encoding gene for resistance to bacterial wilt. AhRRS3 And its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: Firstly, this invention provides a peanut resistance gene against bacterial wilt. AhRRS3 The gene is used in improving resistance to bacterial wilt in plants. AhRRS3 The nucleotide sequence is shown in SEQ ID NO.1.

[0006] Furthermore, the gene AhRRS3 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0007] Furthermore, the plant is peanut or Arabidopsis thaliana.

[0008] Furthermore, overexpression AhRRS3 Genes enhance plant resistance to bacterial wilt.

[0009] Secondly, this invention provides an overexpression of a peanut gene for resistance to bacterial wilt. AhRRS3 Application of expression vectors in improving plant resistance to bacterial wilt, the peanut bacterial wilt resistance gene AhRRS3The nucleotide sequence is shown in SEQ ID NO.1, and the plant is peanut or Arabidopsis thaliana.

[0010] Thirdly, this invention provides a method for improving plant resistance to bacterial wilt, specifically by... AhRRS3 Gene overexpression vectors are used to genetically transform recipient plants, including peanuts and Arabidopsis thaliana. AhRRS3 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] The beneficial effects of this invention are as follows: This invention obtained a high-generation stable recombinant inbred line population (RIL population) through crossbreeding and selection between the disease-resistant peanut variety Yueyou 92 (YY92) and the susceptible variety Xinhui Xiaoli (XHXL). Through multi-year, multi-location planting of the RIL population, resistance was identified by inoculation with Ralstonia solanacearum. The genotype of each RIL line was sequenced, molecular markers were developed, and a high-density genetic map was constructed. Based on the combined identification of the genetic map and the resistance phenotype, QTL fine mapping was used to identify the candidate NBS-LRR gene encoding resistance to bacterial wilt. AhRRS3 The bacterial wilt resistance gene provided by this invention AhRRS3 Overexpression in plants can enhance resistance to bacterial wilt, and under bacterial wilt infection, it can improve the plant's resistance to bacterial wilt, ensuring the normal growth and development of the plant. AhRRS3 The acquisition of disease-resistant genes provides high-quality resistance gene resources for the genetic improvement of peanuts, an important economic crop, and other important crops against bacterial wilt. Attached Figure Description

[0012] Figure 1 The results show the fine mapping of QTLs for resistance to bacterial wilt in peanut recombinant inbred lines (RILs). The candidate gene for resistance to bacterial wilt is located on chromosome 12, Chr12.

[0013] Figure 2 Genes for resistance to bacterial wilt in peanuts AhRRS3 Transient expression of subcellular localization of Nicotiana benthamiana.

[0014] Figure 3 Genes for resistance to bacterial wilt in peanuts AhRRS3 Results of resistance to bacterial wilt in Arabidopsis thaliana transformed with overexpression gene; (A) Phenotypes of plants without Ralstonia solanacearum inoculation; (B) Phenotypes of plants after Ralstonia solanacearum inoculation; (C) Disease index of each plant. The disease index (D) is calculated as follows: D = ∑(Mi×Si)×100 / (N×4), where D represents the disease index; i represents the disease severity level; Mi represents the number of plants with disease severity level i; Si represents the severity level value of disease severity i; and N represents the total number of plants surveyed. Detailed Implementation

[0015] The present invention will be further described below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out in accordance with known means in the art.

[0016] Example 1: Peanut bacterial wilt resistance gene AhRRS3 Construction of overexpression vectors A stable recombinant inbred line (RIL) population was obtained through crossbreeding of the disease-resistant peanut variety Yueyou 92 (YY92) and the susceptible variety Xinhui Xiaoli (XHXL). After years of multi-location planting of the RIL population, resistance was identified by inoculation with Ralstonia solanacearum. Genotypes of each RIL line were sequenced, molecular markers were developed, and a high-density genetic map was constructed. Based on the combined identification of the genetic map and resistance phenotype, QTL fine mapping was used to identify candidate genes for bacterial wilt resistance on chromosome 12 (Chr12). AhRRS3 (Ah12G03440) (nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes as shown in SEQ ID NO.2). Figure 1 The results are for gene localization. This is for cloning from peanuts. AhRRS3 To construct the overexpression vector, the specific primers were designed as follows: AhRRS3-OE-F:5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCTGATGTTGTTTCTGGTG-3' (SEQ ID NO.3); AhRRS3-OE-R:5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTCTTGCTTAAATACTAGTCTCCAATAAGC-3' (SEQ ID NO.4). cDNA from leaves of the disease-resistant variety YY92 was used as a template, and the high-fidelity enzyme PrimeSTAR was used... ® MAX (TAKARA) performed PCR amplification to obtain the disease-resistant varieties. AhRRS3 Gene. The PCR amplification system was: 1 µL cDNA, 2×PrimeSTAR. ®MAX mix 10 µL, forward and reverse primers 0.5 µL each, and pure water added to bring the total reaction volume to 20 µL. PCR reaction conditions were: pre-denaturation 95℃ for 5 min, denaturation 95℃ for 30 s, annealing 55℃ for 30 s, extension 72℃ for 3 min, for a total of 25 cycles. The obtained PCR amplification products were detected by 1% agarose gel electrophoresis. The target band of the correct size was excised, and the PCR product in the gel strip was purified using a gel extraction kit to obtain the target gene. An overexpression vector was constructed using the Gateway system. The target gene was ligated into the pDONR207 vector by Gateway BP enzyme at 25℃ overnight. After the reaction, the ligation product was transformed into competent E. coli cells. After positive colony identification and propagation, positive clones were sent to the company for sequencing to verify the correctness of the recombinant vector. After obtaining the correct sequence, the target gene was ligated into the Pk7WG2.0 overexpression vector using LR ligase.

[0017] Example 2 Peanuts AhRRS3 Transient expression of the gene in *Nicotiana benthamiana* to observe subcellular localization In order to observe AhRRS3 Subcellular localization of the gene-encoded protein was determined, and a gene fusion fluorescent protein expression vector was designed. Specific primers were designed as follows: AhRRS3-YFP-F (5'-tacatttacaattacggatccATGGCTGATGTTGTTTCTGGTGT-3', SEQ ID NO.5) and AhRRS3-YFP-R (5'-ctcgcccttgcccatggatccCTCTTGCTTAAATACTAGTCTCCAATAAGC-3', SEQ ID NO.6). Using leaf cDNA from the disease-resistant variety YY92 as a template, PCR amplification was performed to obtain cDNA without a stop codon. AhRRS3 Gene fragment. PCR amplification system: 2×PrimeSTAR ® 10 µL of MAX mix, 1 µL of cDNA, 0.5 µL each of forward and reverse primers, and add purified water to a total volume of 20 µL. Reaction conditions: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 3 min, for 25 cycles. Restriction endonucleases were used. BamH I digested the vector pFGC-eYFP with enzyme I, and ligated the PCR amplification product to... BamH The recombinant fluorescent expression vector was obtained by cutting the I restriction enzyme site. The fluorescent expression vector was transformed into Agrobacterium competent cells GV3101 using the freeze-thaw method, with the control pFGC-eYFP empty vector set up. The experimental and control groups were injected into Nicotiana benthamiana for transient expression. After 48 hours of light incubation, the yellow fluorescence signal was observed using a fluorescence microscope. The detection results are as follows:Figure 2 visible, AhRRS3 The proteins encoded by the genes are located in the cytoplasm and cell membrane.

[0018] Example 3: Candidate genes for peanut resistance to bacterial wilt AhRRS3 Resistance identification of transgenic Arabidopsis thaliana overexpression In Example 1 AhRRS3 The overexpression vector was transformed into Agrobacterium competent cells GV3101, and positive clones were selected for propagation. The Agrobacterium-mediated transformation was then performed using flower bud infection. AhRRS3 The overexpression vector was introduced into Arabidopsis thaliana Col-0. Positive transgenic plants (OE-AhRRS3#15, OE-AhRRS3#22, OE-AhRRS3#39) were obtained through screening with the antibiotic kanamycin. After sterilization, the harvested plant seeds were inoculated onto 1 / 2 MS medium and transferred to small pots after 10 days. Ralstonia solanacearum was inoculated at the four-to-five-leaf stage (three weeks to four-to-five-leaf stage). Ralstonia solanacearum was streaked onto BG+PB medium and incubated upside down at 28°C for 2 days. Moist, milky-white single colonies from the culture plates were transferred to 1 mL of BG+PB liquid medium and incubated overnight at 28°C with a shaker at 200 rpm. 500 μl of the culture was transferred to 250 mL of BG liquid medium and incubated until the bacterial OD was reached. 600 The concentration was reached to 0.6. After centrifugation and collection of bacterial cells, the bacterial cells were re-selected using sterile water and adjusted to OD. 600 The value was 0.6. A crisscross pattern was made with a blade 1 cm away from the Arabidopsis plant, the depth of which was approximately half the depth of the seedling pot, causing damage to the plant's root system. 5 ml of Ralstonia solanacearum solution was poured into the incision site. Both the experimental group (susceptible plants Col-0 with an overexpression of AhRRS3 transgenic line in the background) and the control group (susceptible plants Col-0 and resistant plants Nd-1) were inoculated with Ralstonia solanacearum. The inoculated Arabidopsis plants were cultured at 28℃ with high temperature and humidity, and the disease incidence was observed daily. The resistant plants Nd-1 and the transgenic resistant varieties... AhRRS3 overexpression lines ( OE-AhRRS3 Both were effective against bacterial wilt, while the infected control group, Col-0, showed signs of complete wilting and death (e.g., ...). Figure 3 The experiment was performed independently three times, and the results were consistent. This proves that overexpression... AhRRS3 Genes can enhance a plant's resistance to bacterial wilt.

[0019] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention shall be covered by the present invention.

Claims

1. Peanut bacterial wilt resistance gene AhRRS3 Its application in improving plant resistance to bacterial wilt is characterized by: The gene AhRRS3 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The gene AhRRS3 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that: The plant in question is Arabidopsis thaliana.

4. The application according to claim 1, characterized in that: overexpression AhRRS3 Genes enhance plant resistance to bacterial wilt.

5. Overexpression of peanut bacterial wilt resistance gene AhRRS3 The application of the expression vector in improving plant resistance to bacterial wilt is characterized by: The peanut gene for resistance to bacterial wilt AhRRS3 The nucleotide sequence is shown in SEQ ID NO.1, and the plant is Arabidopsis thaliana.

6. A method for improving plant resistance to bacterial wilt, characterized in that: Will AhRRS3 Gene overexpression vectors were used to genetically transform recipient plants, wherein the recipient plant was Arabidopsis thaliana. AhRRS3 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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