Peanut anti-rhizoctonia solani nbs-lrr encoding gene ahrrs6 and application thereof

Abstract

The application provides a peanut anti-rhizoctonia solani NBS-LRR encoding gene AhRRS6 and application, the nucleotide sequence of the gene AhRRS6 is as shown in SEQ ID NO.1 (from the genome of a disease-resistant variety YY92) and SEQ ID NO.2 (from the genome of a disease-susceptible variety XHXL). The gene AhRRS6 (SEQ ID NO.1) heterologous overexpression tobacco significantly improves the resistance of tobacco to rhizoctonia solani. By constructing the overexpression vector of AhRRS6 transgenic n. benthamiana, under the infection of rhizoctonia solani, the gene AhRRS6y from the disease-resistant variety is compared with the gene AhRRS6x from the disease-susceptible variety and non-transgenic plants, and the transgenic plants of the disease-resistant variety show obvious disease resistance, while the alleles of the disease-susceptible variety and the non-transgenic control show disease susceptibility. The application provides a gene resource and theoretical basis for the genetic engineering breeding of plant resistance to rhizoctonia solani.

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. AhRRS6 And its applications. Background Technology

[0002] peanut( Arachis hypogaea L. is an important economic oilseed crop worldwide. Bacterial wilt is a disease caused by Ralstonia solanacearum (L.). Ralstonia solanacearum Bacterial wilt is a devastating soil-borne disease affecting a wide range of plants, including eggplant, pepper, tobacco, and tomato, totaling over 450 species. This disease often leads to significant reductions in the yield and quality of peanuts and other important crops. Since biological and agricultural control methods are not very effective against bacterial wilt, identifying resistant genes and breeding resistant varieties is the most effective strategy for controlling the disease. Therefore, the identification and research of genes resistant to bacterial wilt are of great significance and have potential application value in the control of bacterial wilt in peanuts.

[0003] In the early stages of this invention, a high-generation stable (F13) recombinant inbred line (RIL) population was established by crossing the highly resistant variety Yueyou 92 (YY92) with the highly susceptible variety Xinhui Xiaoli (XHXL). More than 500 RIL populations were planted in multiple locations over many years and their resistance phenotypes were identified. Based on this, 30 extremely resistant and 30 extremely susceptible RIL lines were selected to construct mixed pools of resistant and susceptible plants. The resistant parent YY92 and the susceptible parent XHXL were used as controls. The plants of the four groups were inoculated with Ralstonia solanacearum and uninoculated. RNA was extracted from the Ralstonia solanacearum inoculated and uninoculated samples to construct mixed pools of resistant and susceptible RNA with and without inoculation. BSA (Bulk Segregation Analysis) RNA-seq analysis (i.e., BSR-seq) was performed. Allelic SNPs were discovered in the expressed RNA of the parental lines and mixed pools. Using the ΔSNP-index and Euclidean distance algorithms, association analyses were performed on the SNP differences and resistance between the Ralstonia solanacearum-treated and untreated groups, identifying candidate CC-NBS-LRR encoding genes for non-synonymous SNP molecular markers closely linked to resistance. AhRRS6 This provides a resource of resistance genes and markers for improving peanuts and other crops. Summary of the Invention

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

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

[0006] In the early stages, RIL populations were obtained through hybridization of resistant and susceptible varieties, and BSR-seq analysis was performed to identify candidate genes for resistance to bacterial wilt. AhRRS6 In this invention, candidate genes for bacterial wilt resistance were identified by quantitative real-time fluorescence analysis of Ralstonia solanacearum in inoculation of the resistant variety YY92 and the susceptible variety XHXL. AhRRS6 Differences in expression levels. 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.

[0007] This invention provides a peanut NBS-LRR gene encoding resistance to bacterial wilt. AhRRS6 Application of the gene in plant bacterial wilt resistance genetic engineering AhRRS6 The nucleotide sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the plant in question is tobacco.

[0009] Furthermore, overexpression of genes AhRRS6 The gene enhances plant resistance to bacterial wilt. AhRRS6 The nucleotide sequence is shown in SEQ ID NO.1.

[0010] This invention also provides the above-mentioned peanut resistance to bacterial wilt NBS-LRR encoding gene. AhRRS6 The application of the encoded protein in plant bacterial wilt resistance 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.

[0011] This invention also provides an overexpression of the peanut bacterial wilt resistance NBS-LRR encoding gene. AhRRS6 Application of expression vectors 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.

[0012] This invention provides a peanut NBS-LRR encoding gene. AhRRS6 The method for constructing overexpression vectors uses 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 susceptible variety XHXL is shown in SEQ ID NO.2. This gene has no introns, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.4.

[0013] The beneficial effects of this invention are as follows: Peanuts are an important economic oilseed crop, and bacterial wilt disease seriously threatens peanut production. Utilizing genetic engineering technology is a crucial means to address the threat of bacterial wilt to peanut yield; screening for disease-resistant genes and cultivating resistant varieties are the most effective measures for controlling bacterial wilt. Because... AhRRS6 The functional characterization of the gene showed its resistance to bacterial wilt, which can explain AhRRS6 The gene is an effective disease-resistant gene that enhances the plant's defense capabilities when infected by Ralstonia solanacearum, thereby better ensuring the plant's normal growth. The acquisition of this gene provides an important genetic resource for the genetic improvement of peanuts and other crops. Attached Figure Description

[0014] Figure 1 After treating the resistant variety YY92 and the susceptible variety XHXL with Ralstonia solanacearum, candidate genes for resistance to Ralstonia solanacearum were detected by quantitative real-time fluorescence detection at 0 h and 48 h, respectively. AhRRS6 Changes in expression levels.

[0015] Figure 2 Genes for resistance to bacterial wilt in peanuts AhRRS6 Subcellular localization of encoded proteins.

[0016] Figure 3 For the disease-resistant variety YY92 and the susceptible variety XHXL AhRRS6 The results of the alignment of the gene-encoded amino acid sequence.

[0017] Figure 4 For peanuts AhRRS6 Overexpression ( AhRRS6-OE Transgenic tobacco significantly enhances resistance to Ralstonia solanacearum infection. Detailed Implementation

[0018] Example 1: Quantitative fluorescence analysis of Ralstonia solanacearum infection in peanut varieties resistant and susceptible to Ralstonia solanacearum AhRRS6 Gene expression status

[0019] To compare and analyze the effects of Ralstonia solanacearum infection on resistant and susceptible peanut varieties. AhRRS6 Gene expression differences were investigated in peanut varieties YY92 (resistant to bacterial wilt) and XHXL (Xinhui small-grained peanut) treated with Ralstonia solanacearum using the leaf-cutting method. Real-time quantitative PCR was used to examine gene expression at 0h and 48h time points. AhRRS6 Gene-specific primers (AhRRS6-qPCR-F: 5'-GCATTGTCATTAACAAGTCC-3', AhRRS6-qPCR-R: 5'-GTCCAGAAAGTCATCAGCA-3'), and internal reference gene. Ahactin Primers ( Ahactin-qPCR-F: 5'-GAGGAGAATCAGAAGCAAGTC-3', Ahactin -qPCR-R: 5'-CATATACAGCATAGCGGCACTC-3'). Total RNA was extracted from peanut leaves before and after Ralstonia solanacearum infection using the CTAB method. 1 µg of total RNA was reverse transcribed into cDNA using the Novozymes HiScript II 1stStrand cDNA Synthesis Kit. The single-stranded cDNA was diluted 5-fold, and 1 µL of cDNA was used as a template. Real-time quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix (High ROX Premixed) (Vazyme, Nanjing, China) kit. AhRRS6 The relative expression level was determined. A 20 µL reaction system (10 µL 2×SYBR Green buffer, 0.5 µL each of forward and reverse primers, water added to a total volume of 20 µL) was used. Reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 30 s, 40 cycles). The experiment was repeated three times using an ABI 7500 real-time quantitative PCR instrument. 2 -△△Ct Method calculation AhRRS6 The relative expression level of genes, where ΔΔCt = (CT) gene -CT actin ) processing - (CT) gene -CT actin (Control) Student's t-test was used to compare the differences between the control and experimental groups. Results showed that in the resistant variety YY92, after 48 hours of Ralstonia solanacearum infection, AhRRS6 Gene expression levels were significantly increased, while in the susceptible variety XHXL, AhRRS6 There was no significant change in gene expression levels. Figure 1 ).

[0020] Example 2 Peanuts AhRRS6 Construction of gene overexpression vectors

[0021] To build AhRRS6 Gene overexpression vectors, and the design of specific primers:

[0022] AhRRS6-OE-F:5'-AACACGGGGGACTCTTGCCTGCAGGATGGCTAGTGTAGTTGGTGGAGC-3' (SEQ ID NO.5),

[0023] AhRRS6-OE-R:5'-AATGTTTGAACGATCGGGGAAATTCTCAGGAAATTTTGTTGTAATTGACTTC-3' (SEQ ID NO.6);

[0024] cDNA from leaves of the bacterial wilt-resistant variety YY92 and the susceptible variety XHXL was used as templates for PCR amplification, using the high-fidelity enzyme PrimeSTAR from TAKARA. ® MAX amplification, the PCR reaction system was: 1 µL cDNA, 10 µL 2×PrimeSTAR. ® MAX mix, 0.5 µL each of forward and reverse primers, and water to a total volume of 20 µL. Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 3 min, 25 cycles. PCR products were detected by agarose gel electrophoresis and purified by gel excision. Restriction endonucleases... Xba I and Sac The pBI121 vector was double-digested with the following enzymes: 15 μL (3 μg) pBI121 plasmid, 5 μL 10× QuickCut Buffer, 3 μL QuickCut™ XbaI, 3 μL QuickCut™ Sac1, and water to a final volume of 50 μL. Digestion was performed at 37°C for 1 h. The target digested vector was detected by 2% agarose gel electrophoresis and purified by gel excision. Hieff Clone from Shanghai Yisheng Biotechnology Co., Ltd. was used. TM The Multi One Step Cloning Kit is a one-step ligation kit for PCR gene fragments and pBI121 digested vectors. The reaction mixture consists of: 4 μL of 5×CE MultiS Buffer and 50 ng of linearized pBI121 digested vector. AhRRS6 25 ng of gene PCR product, 2 μL of Exnase MultiS, and water were added to a total volume of 20 μL. The ligation reaction was carried out at 37°C for 30 min to obtain the final product. AhRRS6 Overexpression vector. The ligation product was transformed into *E. coli* for identification and sequencing. In resistant and susceptible varieties... RRS6 The gene has no introns; the RRS6 amino acid sequence alignment results for both are as follows: Figure 2 As shown.

[0025] Example 3 Peanuts AhRRS6 Subcellular localization of gene-encoded proteins

[0026] In order to observe AhRRS6Subcellular localization of the gene-encoded protein was determined using primers AhRRS6-YFP-F (5'-tacatttacaattacggatccATGGCTAGTGTAGTTGGTGGAGC-3', SEQ ID NO.7) and AhRRS6-YFP-R (5'-ctcgcccttgcccatggatccGGAAATTTTGTTGTAATTGACTTCAAT-3', SEQ ID NO.8). PCR amplification was performed using cDNA from leaves of the disease-resistant variety YY92 as a template, yielding a protein with a complete reading frame and no stop codon. AhRRS6 PCR products of a gene. Restriction endonuclease. BamH The pFGC-eYFP vector was digested with enzyme I to obtain the target digested vector product. Ligation was performed using the one-step method described in Example 2. AhRRS6 The PCR product of the gene and the pFGC-eYFP restriction enzyme digestion vector yielded a fluorescent expression vector. This vector was transformed into *Agrobacterium tumefaciens* GV3101 via liquid nitrogen freeze-thaw conversion. An empty pFGC-eYFP vector was used as a negative control, and *Nicotiana benthamiana* was injected. After 48 hours of culture under normal light, yellow fluorescence was observed under a fluorescence microscope. The results showed... AhRRS6 The genes encode proteins that are located in the cell membrane and cytoplasm. Figure 3 ).

[0027] Example 4 AhRRS6 Resistance analysis of overexpression transgenic tobacco inoculated with Ralstonia solanacearum

[0028] In Example 2 AhRRS6Overexpression vectors were transformed into Agrobacterium GV3101, and then transformed into Nicotiana benthamiana via Agrobacterium-mediated leaf disc method. Transgenic lines were obtained through kanamycin screening and transgenic identification. Seeds of transgenic plants were sterilized and sown on MS medium containing kanamycin (50 mg / ml). After 10 days, they were transplanted into seedling trays. Two weeks later, uniformly sized transgenic seedlings were selected and transplanted into small pots for further cultivation. After two more weeks, when the seedlings reached the four- to five-leaf stage, they were used for inoculation. Highly pathogenic Ralstonia solanacearum was activated by streaking on TTC medium and incubated upside down at 28°C for 2 days. Single colonies with a pink center and milky white edges were selected and incubated overnight in 1 mL SPA liquid medium (28°C, 200 rpm). 500 μl of the overnight cultured Ralstonia solanacearum was incubated in 250 mL SPA liquid medium until OD600 = 0.6, the cells were collected by centrifugation, resuspended in sterile water, and adjusted to OD600 = 0.6. The root drenching method involves using sterile scissors to make a crisscross pattern in the seedling pot, 1 cm away from the plant, to a depth of half the pot's volume, causing mechanical damage to the plant's root system. Five ml of Ralstonia solanacearum suspension is then applied to each pot at the incision site. Wild-type tobacco (Nb) and transgenic tobacco were inoculated with Ralstonia solanacearum and cultured under high temperature and humidity conditions at 28℃. After 14 days, the disease incidence of the wild-type and transgenic plants was observed. [The text then abruptly shifts to a different topic:] Disease-resistant varieties... AhRRS6 Overexpression transgenic tobacco plants showed significantly enhanced resistance to Ralstonia solanacearum infection compared to wild-type control plants. Wild-type tobacco and susceptible varieties... AhRRS6 Overexpression transgenic plants showed signs of wilting or even death. Figure 4 The experimental treatment was repeated at least three times, and all results showed the same trend, thus proving... AhRRS6 Genes positively regulate the plant's resistance response to bacterial wilt.

[0029] 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 NBS-LRR gene encoding resistance to bacterial wilt AhRRS6 Its application in plant bacterial wilt resistance genetic engineering is characterized by: The gene AhRRS6 The nucleotide sequence is shown in SEQ ID NO.1, indicating the overexpression of the gene. AhRRS6 To improve the resistance of plants to bacterial wilt, the plant being tobacco.

2. The application according to claim 1, characterized in that: The gene AhRRS6 The encoded protein amino acid sequence is shown in SEQ ID NO.

3.

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

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