A tobacco target spot pathogenic factor RsDN3377, a recombinant plasmid, a recombinant bacterial cell, and a preparation method and application of the recombinant bacterial cell

Abstract

The application provides a tobacco target spot pathogen pathogenic factor RsDN3377, a recombinant plasmid, a bacterial body and a preparation method and application of the bacterial body, and belongs to the technical field of biotechnology.The application screens a pathogenic factor generated by tobacco target spot pathogen infection of a host through an RNA-seq method, and reveals important biological functions of the RsDN3377 protein in pathogenicity of target spot pathogen and host interaction.Based on an agrobacterium infiltration transient expression method, a candidate full-length cDNA is constructed into a recombinant plasmid and a recombinant bacterial body, the recombinant bacterial body is injected into a leaf of a benhamiana tobacco N.benthamiana, and necrosis symptoms of a bacterial infection part are observed to identify a molecular function of the RsDN3377 protein.The results show that the agrobacterium infiltration transient expression protein carrying the gene RsDN3377 can cause the occurrence of cell death symptoms, and provides a new target for development and prevention and control of a tobacco target spot pathogen.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a tobacco target spot pathogenic factor RsDN3377, a recombinant plasmid, a recombinant bacterial cell, and a preparation method and application of the recombinant bacterial cell. Background Art

[0002] Rhizoctonia solani is an important pathogenic fungus that can cause diseases in crops such as tobacco, potatoes, tomatoes, and beans. Based on morphological diversity, physiological diversity, host specificity, and pathogenic diversity, R. solani can be divided into at least 14 different fusion groups. Among them, R. solani AG-3PT is the main pathogen of potato black skin disease, which causes wilt and stem rot of potato seedlings. R. solani AG3-TB is an important pathogen that causes tobacco target spot disease, which induces tissue necrosis and perforation damage on leaves, thereby significantly reducing the economic quality of leaves. So far, there are very limited reports on effective fungicides and highly resistant varieties to control the damage of R. solani AG3-TB. Therefore, studying the causative factors of R. solani AG3-TB will provide a valuable theoretical basis for disease control. Summary of the Invention

[0003] In light of this, the present invention aims to provide a virulence factor, RsDN3377, of tobacco target spot pathogen, a recombinant plasmid, a recombinant bacterial cell, and methods for preparing and using the recombinant bacterial cell. Using RNA-seq, the present invention screens for virulence factors produced by tobacco target spot pathogens during host infection, revealing the important biological function of the RsDN3377 protein in the interaction between target spot pathogens and their hosts. This provides a new target for the development and prevention of tobacco target spot disease agents, thereby reducing the threat posed by tobacco target spot disease to its host.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a tobacco target leaf spot pathogenic factor RsDN3377. The amino acid sequence of the tobacco target leaf spot pathogenic factor RsDN3377 is shown in SEQ ID NO: 1.

[0006] Preferably, the nucleotide sequence of the tobacco target spot pathogenicity factor RsDN3377 is shown in SEQ ID NO: 2.

[0007] The invention provides a recombinant plasmid, which comprises the nucleotide sequence of the tobacco target spot pathogenic factor RsDN3377 and an initial vector.

[0008] Preferably, the initial vector is pGR106.

[0009] The present invention provides a recombinant bacterial cell expressing the tobacco target spot pathogenicity factor RsDN3377.

[0010] The present invention provides a method for screening recombinant bacteria, the specific steps of which are as follows:

[0011] (1) Screening for pathogenic proteins secreted by R. solani AG3-TB during infection to obtain gene sequences;

[0012] (2) Design specific primer pairs for the gene sequence and amplify to obtain the target gene fragment;

[0013] (3) Ligating the target fragment with the vector to obtain a ligation product;

[0014] (4) Transforming the ligated product into Escherichia coli and culturing it to obtain the recombinant plasmid;

[0015] (5) The recombinant plasmid is transformed into Agrobacterium competent cells and cultured to obtain recombinant bacteria expressing the pathogenic factor of tobacco target spot pathogen.

[0016] The present invention also provides the use of the tobacco target spot pathogenic factor RsDN3377, the recombinant plasmid and the recombinant bacteria in screening agents against tobacco target spot disease.

[0017] Preferably, the active ingredient of the anti-tobacco target spot disease agent is an inhibitor capable of inhibiting the expression of tobacco target spot disease pathogenic factor RsDN3377 and / or a DNA or RNA sequence capable of reducing the expression level of tobacco target spot disease pathogenic factor RsDN3377.

[0018] The present invention also provides the use of the tobacco target spot pathogenic factor RsDN3377, the recombinant plasmid, and the recombinant bacteria in screening tobacco target spot disease-resistant varieties.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The application adopts the classical secretion pathway (search protein domain) and apoplast and cytoplasm effectors (Effectrop, Apoplast P) to screen pathogenic proteins secreted by R. solani AG3-TB during infection, a total of 807 potential secreted proteins are screened out, based on the RNA-seq sequencing results, 124 apoplast effectors, 236 cytoplasm effectors and 78 potential cysteine-rich small proteins are predicted. Based on the rescreening and amplification, the full length of 10 items is obtained, the relative expression level of the candidate secreted protein is verified by real-time quantitative RT-qPCR, RsDN3377 and RsDN20094 are significantly up-regulated at 48h after inoculation, and the expression amount is up-regulated by 5000 times and 100000 times respectively, RsDN21085, RsDN22904, RsDN22462 and RsDN22450 are up-regulated to the highest at 72h after inoculation. Based on the method of agrobacterium infiltration transient expression, the candidate full-length cDNA is constructed into recombinant plasmid, and is injected into N.benthamiana leaf of Nicotiana benthamiana, and the necrosis symptoms of the inoculation site are observed to identify the molecular function of the candidate protein. Finally, it can be observed that the protein carrying the gene RsDN3377 causes cell death symptoms in the process of agricultural penetration transient expression.

[0021] (2) The RsDN3377 gene obtained in the application can be used to design new pesticide targets in practice according to the structure and function of the gene, and can be used to obtain a persistent resistance variety of target spot disease according to the mutation of the receptor protein gene of RsDN3377 in the host cell. Studying the natural distribution of RsDN3377 gene in the field is beneficial to reveal the composition and variation of physiological races of tobacco target spot fungus, and is helpful for the rational use of disease-resistant varieties and the control of tobacco target spot disease. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The expression pattern of candidate apoplast and cytoplasm effectors, wherein the heat map represents the expression (log 10 RPKM) of apoplast and cytoplasm effector genes, the gene count (>50) screened from Pfam, Blast and reads, the up-regulated cluster is shown in the black box, and the red circle beside the heat map represents the candidate gene determined by RT-qPCR;

[0023] Figure 2The recombinant plasmid was amplified by target fragment and double enzyme digestion (CalI and SalI), wherein A is the amplification of target fragments of RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, RsDN22450, and RsDN21125, and B is the amplification of target fragments of RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, Double enzyme digestion verification of the recombinant plasmids of RsDN22450 and RsDN21125; DN3377 in the figure is RsDN3377, DN21085 is RsDN21085, DN10085 is RsDN10085, DN12825 is RsDN12825, DN20094 is RsDN20094, DN22565 is RsDN22565, DN22904 is RsDN22904, DN22462 is RsDN22462, DN22450 is RsDN22450, and DN21125 is RsDN21125;

[0024] Figure 3 Figure 2 is the expression pattern of 10 candidate secretory protein genes. A is a flow chart of the steps taken for RNA-seq sample preparation and data analysis of tobacco leaves infected with R. solani AG3-TB. B is a flow chart of the 10 genes selected for quantitative real-time RT-qPCR validation at 0 hpi (control), 12 hpi, 48 hpi, and 72 hpi after infection after inoculation with R. solani AG3-TB. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0025] Figure 4 Identification of 10 candidate secretory protein genes induced by agricultural infiltration in N. benthamiana leaves. A shows the expression of 10 putative secretory protein genes. BAX is a mouse pro-apoptotic Bcl-2 family member. GFP construct was used as a negative control, and BAX construct was used as a positive control for infiltration. B shows the observation of leaf cell death by DAB and trypan blue staining. C shows the observation of ROS and necrotic cells in leaves by DAB and trypan blue staining. DETAILED DESCRIPTION

[0026] The present invention provides a tobacco target spot pathogenic factor RsDN3377. The amino acid sequence of the tobacco target spot pathogenic factor RsDN3377 is shown in SEQ ID NO: 1, and is specifically as follows:

[0027] MRFTSLQIATLASALFGLAQASSPLASRATASVVTKCTEDNTVAITFDDGPYTWTTELVDLLDNYGAKGTFFVNGNNYGCIYTEENAERLKDLVKRGHQLASHTWAHAHLPQLTGDALKAEFTRTNEAITKITGRTPAFMRPPYGEYNDEVVETAANNGQTVVIWDFDSQDSIGATAAQSKSYYDNILDNNSGHILTLNHETIETTVHDVIPYALKLIKDKGYKMVTVAQCLGKEPYQATTTAATRDGSWTC.

[0028] In the present application, the nucleotide sequence of the tobacco target alternaria alternata pathogenic factor RsDN3377 is shown as SEQ ID NO: 2, and is specifically as follows:

[0029] .

[0030] The invention provides a recombinant plasmid, which comprises the nucleotide sequence of the tobacco target spot pathogenic factor RsDN3377 and an initial vector.

[0031] In the present invention, the initial vector is pGR106.

[0032] The present invention provides a recombinant bacterial cell expressing the tobacco target spot pathogenicity factor RsDN3377.

[0033] The present invention provides a method for screening recombinant bacteria, the specific steps of which are as follows:

[0034] (1) obtaining a gene sequence by screening pathogenic proteins secreted by R. solani AG3-TB during infection;

[0035] (2) designing a specific primer pair for the gene sequence, amplifying, and obtaining a target gene fragment;

[0036] (3) connecting the target fragment with a vector to obtain a connection product;

[0037] (4) transforming the connection product into E. coli for culture to obtain a recombinant plasmid;

[0038] (5) transforming the recombinant plasmid into an Agrobacterium competent cell for culture to obtain a recombinant bacterium body expressing a tobacco target spot pathogen factor.

[0039] In the present application, a gene sequence is obtained by screening pathogenic proteins secreted by R. solani AG3-TB during infection. The pathogenic proteins secreted by R. solani AG3-TB during infection are screened by using a classical secretion pathway and apoplast and cytoplasm effector to obtain a gene sequence.

[0040] In the present application, a specific primer pair is designed for the gene sequence, amplification is performed, and a target gene fragment is obtained. The specific primer pair is designed for the gene sequence by using a T4 ligase primer design method, and the sequence of the specific primer pair is shown in SEQ ID NO: 23 and SEQ ID NO: 24, specifically, SEQ ID NO: 23, CCATCGATATGCGTTTCACCTCTCTCCAAATTGCC; SEQ ID NO: 24, ACGCGTCGACCTAGCACGTCCAGCTCCCATCACGAGT. The target gene fragment is obtained by amplification, and the reaction system of the amplification is 20 μL, including 1 μL of a template, 0.5 μL of each of 10 μM positive and negative primers, 10 μL of 2×taq Mix, and 7 μL of ddH2O. The reaction conditions of the amplification are as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s; 60℃ annealing for 30 s; 72℃ extension for 1 min (about 350 bp of a fragment, about 1 kb / min), 35 cycles; and 72℃ extension for 8 min.

[0041] In the present invention, the target fragment is ligated to a vector to obtain a ligation product. The target fragment is recombinantly ligated with the pGR106 vector recovered after double enzyme digestion using a T4 ligase DNA Ligation Kit. The enzymes used for double enzyme digestion are Cal I and Sal I. The ligation system comprises: 1 μL of vector fragment at a concentration of 60 ng / μL, 4 μL of target fragment at a concentration of 20 ng / μL-50 ng / μL, and 5 μL of Solution I. Ligation is carried out at 16°C overnight to obtain a ligation product.

[0042] In the present invention, the ligation product is transformed into Escherichia coli and cultured to obtain a recombinant plasmid. The ligation product is transformed into competent Escherichia coli DH5α and cultured. A single E. coli colony grown on a Kan-resistant medium is shaken in LB liquid medium for 6 to 8 hours. PCR and gel electrophoresis are performed using specific primers to determine the band size. The bacterial solution with accurate length is sent for sequencing. The sequencing results are compared using DNAMAN software, and the recombinant plasmid is extracted. The target band is identified by agarose gel electrophoresis and recovered.

[0043] In the present invention, the recombinant plasmid is transformed into Agrobacterium competent cells and cultured to obtain recombinant bacteria expressing the pathogenic factor of tobacco target spot pathogen. The recombinant plasmid is transformed into Agrobacterium competent GV3101 cells by electroporation. The transformation method is as follows: soak the electric shock cup in 75% alcohol for 2 hours, rinse the electric shock cup with distilled water 3 times, rinse with ultrapure water 3 to 8 times, soak in 2mL of anhydrous ethanol for 30 minutes, blow under a clean bench for 20 minutes, and place the electric shock cup at -20°C for standby use; take the Agrobacterium GV3101 competent cells stored at -80°C, melt them in ice for 30 minutes, add 5μL of the recombinant plasmid, mix well, and immediately transfer to the pre-cooled electric shock cup; perform electric shock transformation under the bacterial program; quickly add 80 0 μL of liquid LB was slowly aspirated into a 1.5 mL centrifuge tube, and the cells were shaken at 28° C. for 120 min; the cells were evenly spread on a plate with rifampicin (Rif) and kanamycin (Kan) resistance, and the plates were incubated upside down at 28° C. for 48 h. The cells were picked and transferred to liquid LB with Rif+Kan and shaken for 48 h. Colony PCR and 1% agarose gel electrophoresis were performed for identification. The strains whose identification results met the requirements were shaken. After 48 h, when the bacterial solution turned orange-yellow as a whole, it was centrifuged at 4000 rpm for 10 min, and the cells were recovered to obtain recombinant cells expressing the pathogenic factor of tobacco target spot pathogen.

[0044] The present invention also provides the use of the tobacco target spot pathogenic factor RsDN3377, the recombinant plasmid and the recombinant bacteria in screening agents against tobacco target spot disease.

[0045] In the present application, the active ingredient of the tobacco target spot disease agent is an inhibitor capable of inhibiting the expression of the pathogenic factor RsDN3377 of R. solani and / or a DNA or RNA sequence capable of reducing the expression level of the pathogenic factor RsDN3377 of R. solani.

[0046] The present application also provides the use of the pathogenic factor RsDN3377 of R. solani, the recombinant plasmid and the recombinant bacteria in screening tobacco target spot disease-resistant varieties.

[0047] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0048] The pGR106 vector used in the present application was kindly provided by Professor Sun Xianchao of Southwest University (construction method see Fang, A., Gao, H., Zhang, N., Zheng, X., Sun, X. (2019). A Novel Effector Gene SCRE2 Contributes to Full Virulence of Ustilaginoidea virens to Rice. Front. in Microbiol., 24, 1-16.); E. coli competent cells DH5a and Agrobacterium competent cells GV3101 were purchased from Ugi Biotech Co., Ltd. (Shanghai, China); restriction endonucleases ClaI and SalI were purchased from Baodai Biotech Co., Ltd. (Beijing, China); DL2000, DL15000, DL8000 Maker, Ex taq Mix, dsRNA synthesis kit T7 RNAi Transcription Kit, homologous recombination enzyme were purchased from Novagen Biotech Co., Ltd. (Nanjing, China), T4 ligase was purchased from Baodai Biotech Co., Ltd. (Beijing, China); GFP mouse monoclonal antibody, horseradish peroxidase-labeled mouse secondary antibody were purchased from Biyun Tian Biotech Co., Ltd. (Beijing, China); protein 150kDa Maker, PAGE gel rapid preparation kit, Coomassie brilliant blue rapid destaining solution were purchased from Shanghai Yezhen Biotech Co., Ltd.

[0049] Example 1

[0050] Total RNA was extracted from leaf tissues inoculated with R. solani fungi at different time points (12h, 48h and 72h) using TRIzol reagent (Invitrogen cat. NO. 15596026). The extraction process is as follows:

[0051] 1. Weigh 100 mg of treated leaf tissue and transfer the ground sample into a 1.5 mL pre-chilled clean centrifuge tube using liquid nitrogen. Add 1 mL of TRIzol quickly in a chemical fume hood, vortex immediately, and let the sample stand at room temperature for 5 min after the sample is dispersed;

[0052] 2. Add 200 μL of chloroform at a ratio of 1:0.2, immediately invert the tube 15 times, and let it stand at room temperature for 3 min;

[0053] 3. Centrifuge at 12,000 rpm for 15 min at 4°C. The sample forms three phases after centrifugation. The uppermost water phase contains RNA. Carefully transfer 500 μL of the upper water phase to a 1.5 mL centrifuge tube, and add the same volume of isopropanol. Vortex well and let it stand at room temperature for 10 min;

[0054] 4. Centrifuge at 12,000 rpm for 10 min at 4°C. Discard the supernatant, add 1 mL of 75% ethanol, vortex well, and centrifuge at 7,500 rpm for 5 min at 4°C;

[0055] 5. Discard the supernatant, and place the centrifuge tube on a sterile absorbent paper to absorb the waste liquid. Dry in air for 10-15 min;

[0056] 6. Add 30 μL of ddH2O to resuspend the RNA precipitate, and let it stand on ice for 20 min to dissolve the RNA.

[0057] Prior to mRNA isolation and sequencing, all RNA samples were treated with DNase and then quality was determined using a Nanodrop™ One C Spectrophotometer. The integrity of the RNA was determined using a 1.5% agarose gel electrophoresis and the final qualified RNA was quantified using a Qubit 3.0. Single-stranded RNA sequencing library preparation was performed on total RNA. Each sample was measured in triplicate. The generation sequencing library was constructed following the recommendations of Illumina. Poly(A)-containing mRNA was purified from total RNA using Oligo(dT). The purified mRNA was then fragmented and reverse-transcribed into cDNA. Short fragments were ligated with adapters at both ends. After that, the adapter-ligated cDNA was cleaned up using AxyPrep-Mag PCR clean-up (Axygen) and fragments of about 360 bp were recovered. The product was purified and enriched by PCR (11 cycles) and generated indexed double-stranded cDNA library. The cDNA library was analyzed by Agilent 2100 Bioanalyzer and quantified by Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Subsequently, the library was sequenced by paired-end sequencing under the Illumina HiSeq 6000 (SeqHealth Co., Ltd, Wuhan, China) platform.

[0058] Transcriptomic analysis of R. solani AG3-TB, raw sequencing data were filtered by fastp (version 0.23.0) to discard low-quality reads and trim reads with adapter sequences. Clean and dereplicated data were mapped to the host transcript from https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 000 / 715 / 135 / GCF_000715135.1_Ntab-TN90 / using STAR software (version 2.5.3a) with default parameters. Unmapped reads were reassembled using Trinity default parameters. Sequencing reads were mapped back to the assembled transcripts and the quality of the transcriptome assembly was evaluated using Bowtie2. The longest transcripts of the same gene were selected as unigenes for annotation and DEG analysis. For functional annotation of unigenes, protein databases Nr (NCBI non-redundant protein database), UniProt (Universal Protein Database), Pfam (homologous protein families), eggNog (orthologous groups of genes), GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to infer amino acid sequences.

[0059] The classical secretion pathway (searching protein domain) and apoplast and cytoplasm effectors (Effectrop, Apoplast P) were used to screen the pathogenic proteins secreted by R. solani AG3-TB during infection. A total of 807 potential secreted proteins were screened, and 124 apoplast effectors and 236 cytoplasm effectors were predicted. In addition, small cysteine-rich proteins play a functional role in molecular interactions between fungi and plants, usually with classical structural features, with protein content less than 300 aa and cysteine content more than 4%. A total of 78 potential small cysteine-rich proteins were screened from the R. solani AG3-TB secreted protein data. Figure 1

[0060] The RNA at different inoculation time points was reversely transcribed to synthesize cDNA as a template, and the target gene was amplified by PCR using specific primers. The fragment size was identified by 1% agarose gel electrophoresis. Figure 2 As shown in A in the foregoing table, 10 target gene fragments were obtained by specific primers (F and R) amplification, including: RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, RsDN22450, and RsDN21125. The above 10 target fragments were amplified and were between 300 and 1200 bp. The 10 target fragments were consistent with the theoretical results, and RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, RsDN22450, and RsDN21125 were 759 bp, 594 bp, 414 bp, 342 bp, 312 bp, 351 bp, 342 bp, 558 bp, 1191 bp, and 573 bp, respectively.

[0061] The single colony with a better growth state was picked, and colony PCR was performed to identify the fragment size. The size of the amplified fragment was consistent with the theoretical value by specific primer PCR verification, and was between 300 and 1200 bp. The above-mentioned verified recombinant plasmid was sent to GenScript Biotech Co., Ltd. for sequencing, and the recombinant plasmid with consistent sequencing was verified by double enzyme digestion. Figure 2 ​B) in FIG. 1. The 10 genes were ligated by T4 ligase through the Clal and Sail enzyme digestion sites, and the recombinant plasmid was verified according to the double enzyme digestion. The recombinant plasmid treated by Clal and Sail double enzyme digestion showed two bands, the fragment of about 12000 bp was the vector fragment, and the smaller molecular weight was the gene fragment of interest, which was between 300-1200 bp, consistent with the theoretical value.

[0062] Example 2. RT-qPCR identification of candidate secreted protein expression

[0063] RT-qPCR was performed to identify the 10 candidate secreted proteins obtained in Example 1, to determine the expression levels of the 10 screened secreted proteins in the interaction tissue of R. solani AG3-TB and tobacco. The sample RNA at different inoculation time points (12 h, 48 h and 72 h) was reverse transcribed to synthesize first strand cDNA (Nanjing, China). According to the principle of RT-qPCR primer design, specific primers for candidate genes were designed (Table 1). The expression of candidate genes was identified by RT-qPCR, and the expression levels of RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, RsDN22450, RsDN21125 in the 12 h, 48 h and 72 h samples were analyzed according to the specific primers for RT-qPCR detection.

[0064] Table 1. RT-qPCR primers for candidate secreted proteins

[0065]

[0066]

[0067] Based on RNA-seq sequencing, the expression of candidate differential genes at different infection stages was elucidated, and different potential pathogenic factors in R. solani AG3-TB infection at early stage (6-12 hpi), middle stage (24-36 hpi) and late stage (48-72 hpi) were identified, including toxins, CAZymes, CWDEs and 807 potential secreted proteins and 78 cysteine-rich small proteins Figure 3 A) in FIG. 1. According to the screened potential secreted proteins, 10 candidate pathogenic secreted proteins were determined according to RPKM>100 and Reads>200. The relative expression level of the candidate secreted proteins was verified by real-time quantitative RT-qPCR, and the results are shown in FIG. 2. Figure 3As shown in B, the relative expression levels of the 10 candidate DEGs were up-regulated at 12hpi, 48hpi and 72hpi, among which RsDN3377 and RsDN20094 were significantly up-regulated at 48hpi, with 5000-fold and 100000-fold up-regulation, respectively, and RsDN21085, RsDN22904, RsDN22462 and RsDN22450 were up-regulated to the highest at 72hpi. The results of RT-qPCR were consistent with those of RNA-seq, indicating the accuracy of the results of RNA-seq.

[0068] Example 3 Amplification of candidate gene fragments, construction of recombinant plasmids and transformation of competent cells

[0069] To obtain the candidate protein gene fragments, 10 candidate protein gene full-length fragments were designed according to the T4 ligase primer design method (Table 2). The cDNA of R. solani AG3-TB infected for 12h, 24h and 48h was used as a template, and the full-length of 10 candidate protein genes with restriction sites (RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, RsDN22450, RsDN21125) was amplified according to the PCR reaction. The PCR reaction system was 20μL, containing 1μL of template; 0.5μL of positive and negative primers, respectively; 2xtaq Mix 10μL; ddH2O 7μL. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s; 60℃ annealing for 30s; 72℃ extension for 1min (for fragments about 350bp, about 1kb / min), 35 cycles; 72℃ extension for 8min. The PCR products were identified by running in 1% agarose gel electrophoresis in 1xtAE. To obtain the target fragments, the target fragments were cut and recovered according to the gel recovery method.

[0070] Table 2 Specific primers of candidate genes

[0071]

[0072]

[0073] The pGR106 vector was double-digested with restriction endonucleases (Cal I and Sal I). The total digestion system was 50 μL. The digestion products were confirmed by agarose gel electrophoresis, and the target bands were recovered. The steps were carried out according to the instructions of the product purification kit. The purified PCR product was recombined with the pGR106 vector recovered by double enzyme digestion using the T4 ligase DNA Ligation Kit (Beijing, China). The ligation system contained: 1 μL vector fragment (60 ng / μL), 4 μL target fragment (20 ng / μL-50 ng / μL), and 5 μL Solution I. The ligation was carried out at 16°C overnight. The ligation product was transformed into Escherichia coli competent DH5α and cultured. The transformation method was as follows: E. coli competent DH5α stored at -80°C was thawed on ice; 5 μL of the ligation product was added to 30 μL competent cells, mixed by gently pipetting, and placed on ice for 15 min; the cells were immediately heat-shocked in a 42°C water bath for 45 s, followed by an ice bath for 2 min; 800 μL of LB liquid medium (without antibiotics) was added and cultured on a shaking platform at 37°C to revive the cells for 60 min; the cells were collected, plated on LB solid medium containing Amp resistance, and cultured in an inverted manner at 37°C incubator for 14 h. Single colonies of E. coli grown on resistant medium were picked and shaken in liquid LB with the corresponding antibiotics at 200 rpm for 7 ± 1 h. The culture was then subjected to PCR and identified by electrophoresis on a 1% agarose gel. The culture with consistent fragment size was sent to Sangon Biotech Co., Ltd. for sequencing (Shanghai, China). The sequencing results were compared using DNAMAN, and the plasmids with correct sequencing were extracted. Single colonies of E. coli grown on Kan-resistant medium were shaken in liquid LB medium for 12 h. PCR and gel electrophoresis were performed using specific primers to confirm that the band size was consistent with the expected target fragment size. The culture with the correct length was sent for sequencing (Sangon Biotech Co., Ltd., Shanghai). The sequencing results were compared using DNAMAN software, and the recombinant plasmids were extracted. Finally, 10 recombinant plasmids were obtained: pGR106-DN3377, pGR106-DN21085, pGR106-DN10085, pGR106-DN12825, pGR106-DN20094, pGR106-DN22565, pGR106-DN22904, pGR106-DN22462, pGR106-DN22450, and pGR106-DN21125.

[0074] The recombinant plasmid was transformed into Agrobacterium competent GV3101 cells by electroporation. The specific steps are as follows:

[0075] 1) Clean and disinfect the shock cup in advance by soaking it in 75% alcohol for 2 hours;

[0076] 2) Rinse repeatedly with distilled water three times, rinse with ultrapure water 6-8 times, and soak in 2 mL of anhydrous ethanol for 30 minutes;

[0077] 3) After blowing under a clean bench for 20 minutes, place at -20℃ for later use;

[0078] 4) Take Agrobacterium GV3101 competent cells stored at -80°C, thaw on ice for 30 minutes, add 5 μL of recombinant plasmid, mix well, and immediately transfer to a pre-chilled electroporation cuvette (0.2 cm);

[0079] 5) Perform electroporation transformation under bacterial procedures;

[0080] 6) Quickly add 800 μL of liquid LB to the cuvette, slowly aspirate the LB into a 1.5 mL centrifuge tube, and shake the culture at 28°C for 120 min.

[0081] 7) Evenly spread the bacteria on a plate containing rifampicin (Rif) and kanamycin (Kan) resistance, invert the plate, and incubate in a 28°C incubator for 48 hours. Once the bacteria have grown to a certain size, transfer them to liquid LB containing Rif + Kan and shake for 48 hours. Identify the bacteria by colony PCR and 1% agarose gel electrophoresis.

[0082] 8) Shake a large amount of the above-mentioned strains of uniform size (20 mL). After 48 hours, when the bacterial solution turns orange-yellow, centrifuge at 4000 rpm for 10 minutes to recover the bacteria.

[0083] The cells were suspended in infiltration buffer (preparation method: 4 mL 1 M MES; 4 mL 1 M MgCl2; 400 μL 150 mM AAs diluted to 400 mL sterile distilled water) and the OD was measured using a spectrophotometer. 600 The value is adjusted to keep it within 0.5, and then stand in the dark at room temperature for 2 hours. Use a 1mL syringe to draw the bacterial suspension and inject it into the back of the tobacco leaf. The injection volume for each injection site is 200μL. After injection, culture for 5 days, observe the symptoms and take photos to record the results. Figure 4 .

[0084] Based on the gene expression results of 10 candidate secreted proteins screened from different inoculation time of Example 2, the full length of 10 candidate genes were successfully cloned. Mouse protein BAX, which can induce hypersensitive response in plants overexpressed in inducible plants, was used as a positive control (Wang, Q., Han, C., Ferreira, A. O., Yu, X., Ye, W., Tripathy, S., Kale, S., Gu, B., Sheng, Y., Sui, Y., Wang, X., Zhang, Z., Cheng, B., Dong, S., Shan, W., Zheng, X., Dou, D., Tyler, B., Wang, Y. (2011). Transcriptional programming and functional interactions within the Phytophthora sojae RXLR effector repertoire. Plant Cell, 23, 2064-2086.). To study whether the 10 candidate genes can induce hypersensitive response related to immunity in plants, the recombinant plasmids of candidate full length cDNA were injected into leaves of N. benthamiana based on the method of Agrobacterium infiltration transient expression, and the necrosis symptoms of inoculation sites were observed to identify the molecular function of candidate proteins. The results are shown in Figure 4 As shown in A of FIG. 10, all of the 10 candidate genes were infiltrated into N. benthamiana leaves for 5 days, and only the protein of RsDN3377-carrying gene could induce cell death symptoms in the transient expression of agricultural infiltration. To determine the accumulation of hydrogen peroxide (H2O2) and cell death in Agrobacterium-infiltrated leaves, the inoculation sites were stained, and obvious H2O2 accumulation and cell death were observed at the inoculation sites containing transiently expressed RsDN3377 protein. Figure 4 As shown in B and C of FIG. 10, compared with the negative GFP control, RsDN3377 accumulated more H2O2 and induced cell death.

[0085] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A tobacco target spot pathogenic factor RsDN3377 gene, characterized in that: The amino acid sequence encoded by the tobacco target leaf spot pathogenic factor RsDN3377 gene is shown in SEQ ID NO:

1.

2. The tobacco target spot pathogenicity factor RsDN3377 gene according to claim 1, characterized in that The nucleotide sequence of the tobacco target leaf spot pathogenic factor RsDN3377 gene is shown in SEQ ID NO:

2.

3. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleotide sequence of the tobacco target spot pathogenic factor RsDN3377 gene according to claim 2 and an initial vector.

4. The recombinant plasmid according to claim 3, characterized in that The original vector was pGR106.

5. A recombinant bacterial cell expressing the tobacco target spot pathogenicity factor RsDN3377 gene according to claim 1.

6. Use of the tobacco target spot pathogenicity factor RsDN3377 gene according to claim 1 or 2, the recombinant plasmid according to claim 3 or 4, and the recombinant bacteria according to claim 5 in screening agents against tobacco target spot disease, characterized in that: The anti-tobacco target spot agent is an agent that inhibits the expression of the RsDN3377 gene.

7. The use according to claim 6, characterized in that The active ingredient of the anti-tobacco target spot disease agent is an inhibitor capable of inhibiting the expression of the tobacco target spot disease pathogenic factor RsDN3377 gene and / or a DNA or RNA sequence capable of reducing the expression level of the tobacco target spot disease pathogenic factor RsDN3377 gene.

8. Use of the tobacco target spot pathogenicity factor RsDN3377 gene according to claim 1 or 2, the recombinant plasmid according to claim 3 or 4, and the recombinant bacterium according to claim 5 in screening tobacco target spot disease-resistant varieties, characterized in that: The tobacco target spot disease-resistant variety is a variety that suppresses the expression of the RsDN3377 gene.

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