Peanut bacterial wilt resistant NBS-LRR coding gene AhRRS6 and application thereof

Abstract

The invention provides a peanut bacterial wilt resistant NBS-LRR coding gene AhRRS6 and application thereof. Nucleotide sequences of the gene AhRRS6 are shown as SEQ ID NO.1 (from a genome of a disease resistant variety YY92) and SEQ ID NO.2 (from a genome of a susceptible variety XHXL). The gene AhRRS6 (SEQ ID NO.1) is used for heterologous overexpression of tobaccos, so that the resistance of the tobaccos to bacterial wilt is remarkably improved. An overexpression vector of AhRRS6 is constructed to transgenosis Bensi tobacco, under infection of ralstonia solanacearum, AhRRS6y from a disease-resistant variety is compared with an AhRRS6x gene of a susceptible variety and a non-transgenic plant, the transgenic plant of the disease-resistant variety shows obvious disease resistance, and allelic genes of the susceptible variety and non-transgenic control show susceptibility. The invention provides a gene resource and a theoretical basis for genetic engineering breeding of plant bacterial wilt resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a peanut NBS-LRR coding gene for resistance to bacterial wilt. AhRRS6 and its applications. Background Art

[0002] peanut( Arachis hypogaea L.) is an important economic oil crop in the world. Bacterial wilt is a disease caused by Ralstonia solanacearum ( Ralstonia solanacearum ) is a soil-borne devastating disease that affects a wide range of hosts, including more than 450 plant species such as eggplant, pepper, tobacco, and tomato. This disease often leads to a significant reduction in the yield and quality of peanuts and other important crops. Since biological and agricultural control methods are not effective against bacterial wilt, the identification of resistance genes and the breeding of resistant varieties are the most effective strategies for the prevention and control of bacterial wilt. Therefore, the identification and research of genes resistant to bacterial wilt are of great significance and potential application value in the prevention and control of peanut bacterial wilt.

[0003] In the early stage of the present invention, a high-generation stable (F13) recombinant inbred line (RIL) population was established by hybridizing a highly resistant variety Yueyou 92 (YY92) with a highly susceptible variety Xinhui Xiaoli (XHXL), and more than 500 RIL populations were planted for many years and identified for resistance phenotypes. On this basis, 30 extremely highly resistant and extremely highly susceptible RIL lines were selected, and disease-resistant and disease-susceptible mixed pools were constructed. The disease-resistant parent YY92 and the disease-susceptible parent XHXL were used as controls. Four groups of material plants were inoculated with and not inoculated with Ralstonia solanacearum, respectively. RNA was extracted from samples inoculated with and not inoculated with Ralstonia solanacearum, and mixed pools of resistant and susceptible RNA with and without inoculation were constructed, and BSA (Bulk Segregation Analysis) RNA-seq analysis (i.e., BSR-seq) was performed. Allele SNP mining of RNA expressed in parents and mixed pools was performed. The association analysis between SNP differences and resistance between Ralstonia solanacearum treated and untreated groups was performed using ΔSNP-index and Euclidean distance algorithms, and candidate CC-NBS-LRR coding genes of non-synonymous SNP molecular markers closely linked to resistance were identified. AhRRS6 , providing resistance gene and marker resources for peanut and other crop improvements. Summary of the invention

[0004] The purpose of the present invention is to provide a peanut bacterial wilt resistance NBS-LRR encoding gene AhRRS6 and its applications.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: In the early stage, the RIL population was obtained by hybridizing resistant and susceptible varieties and BSR-seq analysis was performed to obtain candidate genes for resistance to bacterial wilt AhRRS6 In the present invention, the resistant variety YY92 and the susceptible variety XHXL were inoculated with Ralstonia solanacearum to quantitatively identify candidate genes for resistance to bacterial wilt. AhRRS6 The expression level difference of AhRRS6 The gene was fused with the fluorescent protein expression tag YFP and transiently expressed in tobacco to observe the subcellular localization of the protein encoded by the disease resistance gene.

[0006] The present invention provides a peanut bacterial wilt resistance NBS-LRR encoding gene AhRRS6 Application of the gene in plant resistance to bacterial wilt genetic engineering, the gene AhRRS6 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] Furthermore, the plant is tobacco.

[0008] Furthermore, overexpressed genes AhRRS6 Improving plant resistance to bacterial wilt, the gene AhRRS6 The nucleotide sequence is shown in SEQ ID NO.1.

[0009] The present invention also provides the above-mentioned peanut bacterial wilt resistance NBS-LRR encoding gene AhRRS6 Application of the encoded protein in plant resistance to bacterial wilt genetic engineering, the gene AhRRS6 The amino acid sequence of the protein encoded in the disease-resistant variety is shown in SEQ ID NO.3, and the plant is tobacco.

[0010] The present invention also provides a method for overexpressing the NBS-LRR coding gene for peanut resistance to bacterial wilt. AhRRS6 Application of an expression vector in plant bacterial wilt resistance gene engineering, the peanut bacterial wilt resistance gene AhRRS6 The nucleotide sequence is shown in SEQ ID NO.1, and the plant is tobacco.

[0011] The present invention provides a method comprising the peanut NBS-LRR encoding gene AhRRS6 Method for constructing an overexpression vector using the CaMV 35S overexpression promoter AhRRS6 The gene AhRRS6 The nucleotide sequence in the disease-resistant variety YY92 is shown in SEQ ID NO.1; the sequence in the disease-susceptible variety XHXL is shown in SEQ ID NO.2. The gene has no introns, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.4.

[0012] The beneficial effects of the present invention are as follows: peanuts are important economic oil crops, and bacterial wilt disease seriously threatens the production of peanuts. The use of genetic engineering technology is an important means to solve the threat of bacterial wilt disease to peanut production. Screening for disease-resistant genes and cultivating resistant varieties are the most effective measures to prevent and control bacterial wilt disease. AhRRS6 Functional characterization of the gene showed resistance to bacterial wilt, which could explain AhRRS6 The gene is an effective disease-resistant gene that can improve the plant's defense ability when infected by Ralstonia solanacearum, thereby better ensuring the normal growth of the plant. The acquisition of this gene provides an important genetic resource for the genetic improvement of peanuts and other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Fluorescence quantitative detection of candidate genes for resistance to bacterial wilt in the resistant variety YY92 and the susceptible variety XHXL at 0 h and 48 h after treatment with Ralstonia solanacearum AhRRS6 changes in expression levels.

[0014] Figure 2 Peanut bacterial wilt resistance gene AhRRS6 Subcellular localization of the encoded protein.

[0015] Figure 3 It is the disease-resistant variety YY92 and the susceptible variety XHXL AhRRS6 Comparison results of gene-encoded amino acid sequences.

[0016] Figure 4 For Peanuts AhRRS6 Overexpression ( AhRRS6-OE ) Transgenic tobacco has significantly enhanced resistance to infection by Ralstonia solanacearum. DETAILED DESCRIPTION

[0017] Example 1 Fluorescence quantitative analysis of Ralstonia solanacearum infection in resistant and susceptible peanut varieties AhRRS6 Gene expression To compare and analyze the effects of peanut varieties resistant and susceptible to bacterial wilt infection on AhRRS6 Gene expression differences were detected by real-time fluorescence quantitative PCR at 0h and 48h after the peanut varieties Yueyou 92 (YY92) and Xinhui Xiaoli (XHXL) were inoculated with Ralstonia solanacearum by leaf clipping method. AhRRS6 Gene-specific primers (AhRRS6-qPCR-F: 5'-GCATTGTCATTAACAAGTCC-3', AhRRS6-qPCR-R: 5'-GTCCAGAAAGTCATCAGCA-3'), and internal reference genes Ahactin Primer ( Ahactin -qPCR-F: 5'-GAGGAGAATCAGAAGCAAGTC-3',Ahactin -qPCR-R: 5'-CATATACAGCATAGCGGCACTC-3'). The total RNA of peanut leaves before and after Ralstonia solanacearum infection was extracted by CTAB method, and 1 µg of total RNA was reverse transcribed into cDNA using HiScript II 1stStrand cDNA Synthesis Kit of Novazon Biotech Co., Ltd. The single-stranded cDNA was diluted 5 times, and 1 µL of cDNA was taken as template. Real-time fluorescence quantitative PCR detection was performed using ChamQ SYBR qPCR Master Mix (High ROX Premixed) (Vazyme, Nanjing, China) kit method. AhRRS6 The relative expression level of 20μL reaction system (10 μL 2×SYBR Green buffer, 0.5 μL each of forward and reverse primers, add water to a total volume of 20μL, reaction conditions (95℃ pre-denaturation for 5 min; 95℃ 15 s, 60℃ 15 s, 72℃ 30 s, 40 cycles). The experiment was repeated three times using ABI7500 real-time fluorescence quantitative PCR instrument. 2 -△△Ct Calculation AhRRS6 The relative expression level of the gene, where △△Ct= (CT gene -CT actin )Processing-(CT gene -CT actin ) control. The Student's t test was used to compare the differences between the control group and the experimental group. The results showed that in the disease-resistant variety YY92, after 48 hours of infection with Ralstonia solanacearum, AhRRS6 The gene expression level was significantly increased, and in the susceptible variety XHXL, AhRRS6 There was no significant change in gene expression ( Figure 1 ).

[0018] Example 2 Peanuts AhRRS6 Construction of gene overexpression vector To build AhRRS6 Gene overexpression vector, design specific primers: AhRRS6-OE-F:5'-AACACGGGGGACTCTTGCCTGCAGGATGGCTAGTGTAGTTGGTGGAGC-3' (SEQ ID NO.5), AhRRS6-OE-R:5'-AATGTTTGAACGATCGGGGAAATTCTCAGGAAATTTTGTTGTAATTGACTTC-3' (SEQ ID NO.6); The cDNA of the leaves of the bacterial wilt-resistant variety YY92 and the susceptible variety XHXL were used as templates for PCR amplification, using the high-fidelity enzyme PrimeSTAR from TAKARA. ® MAX amplification, PCR reaction system: 1 µL cDNA, 10 µL 2× PrimeSTAR ® MAX mix, 0.5 µL each of forward and reverse primers, add water to a total volume of 20 µL. Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ 30 s, 55℃ 30 s, 72℃ 3 min, 25 cycles. Detection by agarose gel electrophoresis and gel purification to recover PCR products. Restriction endonuclease Xba I and Sac I double-digested pBI121 vector, the digestion conditions were: pBI121 plasmid 15 μL (3 μg), 10×QuickCut Buffer 5 μL, QuickCut™ XbaI 3 μL, QuickCut™ Sac1 3 μL, add water to 50 μL, and digest at 37℃ for 1 h. 2% agarose gel electrophoresis was used to detect and purify the target digested vector. Hieff Clone from Shanghai Yisheng Biotechnology Co., Ltd. was used. TM Multi One Step Cloning Kit, one-step ligation of PCR gene fragments and pBI121 restriction vector, reaction system: 5×CE MultiS Buffer 4 μL, linearized pBI121 restriction vector 50ng, AhRRS6 25 ng of gene PCR product, 2 μL of Exnase MultiS, add water to a total volume of 20 μL, and react at 37°C for 30 min to obtain AhRRS6 Overexpression vector. The ligation product was transformed into Escherichia coli for identification and sequencing. RRS6 The gene has no introns. The comparison results of the amino acid sequences of RRS6 of the two are as follows: Figure 2 shown.

[0019] Example 3 Peanut AhRRS6 Subcellular localization of gene-encoded proteins To observe AhRRS6To investigate the subcellular localization of the protein encoded by the gene, primers AhRRS6-YFP-F (5'-tacatttacaattacggatccATGGCTAGTGTAGTTGGTGGAGC-3', SEQ ID NO.7) and AhRRS6-YFP-R (5'-ctcgcccttgcccatggatccGGAAATTTTGTTGTAATTGACTTCAAT-3', SEQ ID NO.8) were designed and PCR amplified using cDNA from the leaves of the disease-resistant variety YY92 as a template. AhRRS6 PCR products of genes. Restriction endonucleases BamH I digested the pFGC-eYFP vector to obtain the target digested vector product. The one-step ligation method in Example 2 was used. AhRRS6 The PCR product of the gene and the pFGC-eYFP restriction vector were cut to obtain the fluorescent expression vector. The fluorescent expression vector was transformed into Agrobacterium GV3101 by liquid nitrogen freeze-thaw method, and the pFGC-eYFP empty vector was used as a negative control and injected into Nicotiana benthamiana. After 48 hours of normal light culture, yellow fluorescence was observed under a fluorescence microscope. The observation results showed AhRRS6 The protein encoded by the gene is located in the cell membrane and cytoplasm ( Figure 3 ).

[0020] Example 4 AhRRS6 Resistance analysis of overexpression transgenic tobacco inoculated with Ralstonia solanacearum In Example 2 AhRRS6Agrobacterium GV3101 was transformed with the overexpression vector, and Nicotiana benthamiana was transformed by the leaf disc method mediated by Agrobacterium tumefaciens. Transgenic lines were obtained by kanamycin screening and transgenic identification. The seeds of transgenic plants were sterilized and sown on MS medium containing kanamycin (50 mg / ml). After 10 days, they were transplanted to seedling trays. Two weeks later, transgenic seedlings of the same size were selected and transplanted to small pots for inoculation after two weeks of cultivation. The highly pathogenic Ralstonia solanacearum was activated by streaking on TTC medium, incubated in a constant temperature incubator at 28°C for 2 days, and a single clone with pink in the center and milky white on the edge was picked and cultured overnight in 1 mL SPA liquid medium (28°C, 200 rpm). Take 500μl of the overnight cultured Ralstonia solanacearum and culture it in 250ml SPA liquid medium until OD600=0.6, collect the cells by centrifugation, resuspend with sterile water and make OD600=0.6. The root irrigation method was used, that is, a "well" shape was cut in a small pot 1 cm away from the plant with a sterile scissors blade, and the depth was 1 / 2 of the seedling pot, causing mechanical damage to the root system of the plant. 5 ml of the solanacearum suspension was poured into each pot of plants at the cut edge. The wild-type tobacco Nb and transgenic tobacco were inoculated with solanacearum, respectively, and cultured under high temperature and high humidity conditions at 28°C. After 14 days, the disease of the wild-type and transgenic plants was observed. AhRRS6 The overexpression transgenic tobacco plants showed significantly enhanced resistance to Ralstonia solanacearum infection compared to the wild-type plants. AhRRS6 Overexpressing transgenic plants showed wilting or even death ( Figure 4 The experimental treatment was repeated at least three times, and the results showed the same trend, which proved AhRRS6 The gene positively regulates the plant's resistance response to bacterial wilt.

[0021] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. NBS-LRR encoding gene for peanut resistance to bacterial wilt AhRRS6 The application in plant resistance to bacterial wilt genetic engineering is characterized by: The gene AhRRS6 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The peanut bacterial wilt resistance NBS-LRR encoding gene according to claim 1 AhRRS6 The application in plant resistance to bacterial wilt genetic engineering is characterized by: The plant is tobacco.

3. The peanut bacterial wilt resistance NBS-LRR encoding gene according to claim 1 AhRRS6 The application in plant resistance to bacterial wilt genetic engineering is characterized by: Overexpressed genes AhRRS6 Improving plant resistance to bacterial wilt, the gene AhRRS6 The nucleotide sequence is shown in SEQ ID NO.

1.

4. The peanut bacterial wilt resistance NBS-LRR encoding gene according to claim 1 AhRRS6 The application of the encoded protein in plant resistance to bacterial wilt genetic engineering is characterized by: The gene AhRRS6 The amino acid sequence of the encoded protein is shown in SEQ ID NO.3, and the plant is tobacco.

5. Overexpression of NBS-LRR encoding genes for peanut resistance to bacterial wilt AhRRS6 The use of an expression vector in plant resistance to bacterial wilt gene engineering is characterized by: The peanut bacterial wilt resistance gene AhRRS6 The nucleotide sequence is shown in SEQ ID NO.1, and the plant is tobacco.

Images