Tobacco target leaf pathogen pathogenic factor RsDN3377, recombinant plasmid, recombinant thallus and preparation method and application of recombinant thallus

Abstract

The invention provides a pathogenic factor RsDN3377 of tobacco target leaf spot pathogen, a recombinant plasmid, a thallus and a preparation method and application of the thallus, and belongs to the technical field of biology. According to the invention, pathogenic factors generated by tobacco target leaf spot pathogen infected hosts are screened by an RNA-seq method, and the important biological function of the RsDN3377 protein in the interaction of target leaf spot pathogen and hosts is disclosed. The candidate full-length cDNA is used for constructing recombinant plasmids and recombinant thalli based on an agrobacterium infiltration transient expression method, the recombinant thalli are injected to nicotiana benthamiana N.benthamiana leaves, and the necrosis symptom of the inoculation part is observed to identify the molecular function of the RsDN3377 protein. The result shows that the protein which carries the gene RsDN3377 and is infiltrated and transiently expressed by the agrobacterium can cause the occurrence of a cell death symptom, and a new target spot is provided for medicament development and prevention and control of the tobacco target leaf spot.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly 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, potato, tomato, 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 potato seedling wilt and stem rot. 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 view of this, the purpose of the present invention is to provide 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. The present invention screens the pathogenic factors produced by tobacco target spot pathogens infecting hosts by RNA-seq method, and reveals the important biological function of RsDN3377 protein in the interaction between target spot pathogens and hosts. It provides new targets for the development and prevention of tobacco target spot disease drugs, so as to reduce the threat of tobacco target spot disease to hosts.

[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 spot pathogenicity factor RsDN3377, and the amino acid sequence of the tobacco target spot pathogenicity factor RsDN3377 is shown in SEQ ID NO:1.

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

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

[0008] Preferably, the initial vector is pGR106.

[0009] The invention provides a recombinant bacterial body 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) Obtaining gene sequences by screening pathogenic proteins secreted by R.solaniAG3-TB during infection;

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

[0013] (3) Connecting the target fragment to the vector to obtain a connection product;

[0014] (4) Transforming the ligated product into Escherichia coli and culturing it to obtain a 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 anti-tobacco target spot disease agents.

[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 pathogenicity factor RsDN3377 and / or a DNA or RNA sequence capable of reducing the expression level of tobacco target spot disease pathogenicity factor RsDN3377.

[0018] The present invention also provides the application 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 present invention uses the classical secretion pathway (searching protein domains) and apoplast and cytoplasm effectors (Effectrop, ApoplastP) to screen the pathogenic proteins secreted by R.solani AG3-TB during infection, and screens out a total of 807 potential secretory proteins. Based on the RNA-seq sequencing results, 124 apoplast effectors, 236 cytoplasm effectors and 78 potential cysteine-rich small proteins were predicted. Based on the full-length fragments of 10 entries obtained by multiple screening and amplification, the relative expression levels of the candidate secretory proteins were verified by real-time quantitative RT-qPCR. The expression levels of RsDN3377 and RsDN20094 were significantly upregulated at 48h of inoculation, upregulated by 5000 times and 100000 times, respectively. The expression levels of RsDN21085, RsDN22904, RsDN22462, and RsDN22450 were upregulated to the highest level at 72h of inoculation. Based on the method of Agrobacterium infiltration transient expression, the candidate full-length cDNA construct recombinant plasmid was injected into the leaves of N. benthamiana, and the necrotic symptoms at the inoculated site were observed to identify the molecular function of the candidate protein. Finally, it was observed that the protein transiently expressed by the agricultural infiltration carrying the gene RsDN3377 caused cell death symptoms.

[0021] (2) The RsDN3377 gene obtained by the present invention can be used in practice to design new pesticide targets based on the structure and function of the gene, and can obtain durable resistant varieties to target spot disease based on the mutation of the receptor protein gene of RsDN3377 in host cells. Studying the natural distribution of the RsDN3377 gene in the field is conducive to revealing the composition and variation of the physiological subspecies of tobacco target spot pathogens, which is helpful for the rational use of disease-resistant varieties and controlling the occurrence of tobacco target spot disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The expression patterns of candidate apoplast and cytoplasm effectors are shown in the heatmap, where the expression of apoplast and cytoplasm effector genes (log 10 RPKM), gene counts (>50) screened from Pfam, Blast, and reads, upregulated clusters are shown in dotted black boxes, and red circles next to the heat map represent candidate genes determined by RT-qPCR;

[0023] Figure 2The recombinant plasmid was identified by double enzyme digestion (CalI and SalI), wherein A is the amplification of the target fragment of RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, RsDN22450, and RsDN21125, and B is RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, Double enzyme digestion verification of the recombinant plasmids of RsDN22450 and RsDN21125; in the figure, DN3377 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 is the expression pattern of 10 candidate secretory protein genes, where 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, and B is a total of 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 leaf cell death, where A is 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 is the observation of leaf cell death by DAB and trypan blue staining, and C is the observation of ROS and necrotic cells in leaves using DAB and trypan blue staining. DETAILED DESCRIPTION

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

[0027] MRFTSLQIATLASALFGLAQASSPLASRATASVVTKCTEDNTVAITFDDGPYTWTTELVDLLDNYGAKGTFFVNGNNYGCIYTEENAERLKDLVKRGHQLASHTWAHAHLPQLTGDALKAEFTRTN EAITKITGRTPAFMRPPYGEYNDEVVETAANNGQTVVIWDFDSQDSIGATAAQSKSYYDNILDNNSGHILTLNHETIETTVHDVIPYALKLIKDKGYKMVTVAQCLGKEPYQATTTAATRDGSWTC.

[0028] In the present invention, the nucleotide sequence of the tobacco target spot pathogenic factor RsDN3377 is shown in SEQ ID NO: 2, which is as follows:

[0029] .

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

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

[0032] The invention provides a recombinant bacterial body 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 gene sequences by screening pathogenic proteins secreted by R.solaniAG3-TB during infection;

[0035] (2) Designing specific primer pairs for the gene sequence and amplifying to obtain the target gene fragment;

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

[0037] (4) Transforming the ligated product into Escherichia coli and culturing it to obtain a recombinant plasmid;

[0038] (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.

[0039] In the present invention, the pathogenic proteins secreted by R.solaniAG3-TB during infection are screened to obtain the gene sequence. The pathogenic proteins secreted by R.solaniAG3-TB during infection are screened using the classical secretion pathway and apoplast and cytoplasm effectors to obtain the gene sequence.

[0040] In the present invention, a specific primer pair is designed for the gene sequence, and amplification is performed to obtain the target gene fragment. A specific primer pair is designed for the gene sequence by the 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; amplification is performed to obtain the target gene fragment, and the reaction system of the amplification is calculated in 20 μL: 1 μL of template; 0.5 μL of each of 10 μM positive and negative primers; 10 μL of 2×taq Mix; 7 μL of ddH2O, and the reaction conditions of the amplification are: 95°C pre-denaturation for 5 min; 94°C denaturation for 30 s; 60°C annealing for 30 s; 72°C extension for 1 min (fragment length is about 350 bp, about 1 kb / min), 35 cycles; 72°C extension for 8 min.

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

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

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

[0045] In the present invention, 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.

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

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] The pGR106 vector used in the present invention was donated by Professor Sun Xianchao of Southwest University (for the construction method, see Fang, A., Gao, H., Zhang, N., Zheng, X., Sun, X. (2019). A Novel Effector Gene SCRE2 Contributes to Full Virulence of Ustilaginoidevirens to Rice. Front. in Microbiol., 24, 1-16.); Escherichia coli competent DH5α and Agrobacterium competent cells GV3101 were purchased from Weidi Biotechnology Co., Ltd. (Shanghai, China); restriction endonucleases ClaΙ and SalΙ were purchased from Baoriyi Biotechnology Co., Ltd. (Beijing, China); DL2000, DL15000, DL8000 Maker, Ex taq Mix, dsRNA synthesis kit T7 RNAi Transcription Kit, homologous recombination enzyme was purchased from Novozymes Biotech Co., Ltd. (Nanjing, China), T4 ligase was purchased from Bioray Biotech Co., Ltd. (Beijing, China); GFP mouse monoclonal antibody and horseradish peroxide-labeled mouse secondary antibody were purchased from Biotechnology Co., Ltd. (Beijing, China); protein 150 kDa Maker, PAGE gel rapid preparation kit, and Coomassie brilliant blue rapid decolorization-free stain were purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.

[0049] Example 1

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

[0051] 1. Weigh 100 mg of the treated leaf tissue, grind the sample thoroughly with liquid nitrogen and transfer it to a 1.5 mL pre-cooled clean centrifuge tube. Quickly add 1 mL of TRIzol in a chemical fume hood, vortex and shake quickly to break up the sample and place it at room temperature for 5 minutes.

[0052] 2. Add 200 μL of chloroform in a ratio of 1:0.2, immediately invert for 15 seconds, and leave at room temperature for 3 minutes;

[0053] 3. Centrifuge at 12000rpm for 15min at 4℃. The sample after centrifugation forms a three-phase water system. The uppermost water phase contains RNA. Carefully transfer 500μL of the upper water phase to a 1.5mL centrifuge tube and add the same volume of isopropanol. Vortex and shake thoroughly to mix and let stand at room temperature for 10min.

[0054] 4. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, add 1 mL of 75% ethanol, vortex to mix, and centrifuge at 7500 rpm for 5 min at 4°C;

[0055] 5. Discard the supernatant, place the centrifuge tube on sterilized absorbent paper to fully absorb the waste residual liquid, and dry in the air for 10 to 15 minutes;

[0056] 6. Add 30 μL ddH2O to resuspend the RNA pellet and place on ice for 20 minutes to fully dissolve the RNA.

[0057] Prior to mRNA isolation and sequencing, all RNA samples were treated with DNase and then the quality was determined using a NanodropTM OneC spectrophotometer. 1.5% agarose gel electrophoresis was used to determine the integrity of the RNA, and Qubit 3.0 was used to quantify the final qualified RNA. Single-stranded RNA sequencing library preparation was performed on total RNA. Each sample was measured three times. The next generation sequencing library was constructed according to Illumina's recommendations. Oligo(dT) was used to purify poly(A)-containing mRNA from total RNA. The purified mRNA was then fragmented and reverse transcribed into cDNA. The short fragments were connected at both ends with adapters. Thereafter, AxyPrep-Mag PCR cleanup (Axygen) was used for adapter-ligated cDNA, and fragments of approximately 360 bp were recovered. The products were purified and enriched by PCR (11 cycles), and an indexed double-stranded cDNA library was generated. 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 on the Illumina HiSeq 6000 (SeqHealth Co., Ltd, Wuhan, China) platform.

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

[0059] The classical secretion pathway (searching for protein domains) as well as apoplast and cytoplasm effectors (Effectrop, ApoplastP) were used to screen for pathogenic proteins secreted by R.solaniAG3-TB during infection. A total of 807 potential secreted proteins were screened, and 124 apoplast effectors and 236 cytoplasm effectors were predicted. In addition, cysteine-rich small proteins play functional roles in molecular interactions between fungi and plants, and usually have classic structural characteristics, with a protein content of less than 300aa and a cysteine ​​content of more than 4%. 78 potential cysteine-rich small proteins were screened from the R.solaniAG3-TB secreted protein data ( Figure 1 ).

[0060] The cDNA synthesized by reverse transcription of RNA at different inoculation time points was used as a template, and the target gene was amplified by PCR using specific primers, and the fragment size was identified under 1% agarose gel electrophoresis. Figure 2 As can be seen from A, 10 gene fragments were amplified by specific primers (F and R), including: RsDN3377, RsDN21085, RsDN10085, RsDN12825, RsDN20094, RsDN22565, RsDN22904, RsDN22462, RsDN22450, and RsDN21125. The fragments of the above 10 items were all amplified, and were all between 300 and 1200 bp. The fragments of the 10 items were consistent with the theoretical results. The fragments of 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] Single colonies with better growth status were selected, and colony PCR was performed on the single colonies to identify the fragment size under 1% agarose gel electrophoresis. The size of the amplified fragment obtained by PCR with specific primers was consistent with its theoretical value, both between 300 and 1200 bp. The recombinant plasmids successfully verified above were sent to Sangon Biotech Co., Ltd. for sequencing, and the recombinant plasmids with consistent sequencing were verified by double enzyme digestion ( Figure 2B). The genes of 10 items were connected by T4 ligase through ClaI and Sal I restriction sites, and the recombinant plasmid was verified by double enzyme digestion. The recombinant plasmid treated with ClaI and SalI double enzyme digestion showed two obvious bands. The fragment of about 12000bp was the vector fragment, and the smaller molecular weight was the target gene fragment. Both were between 300 and 1200bp, which was consistent with the theoretical value.

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

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

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

[0065]

[0066]

[0067] Based on RNA-seq sequencing, we elucidated the expression of candidate differentially expressed genes at different infection stages and identified different potential pathogenic factors in the early (6-12 hpi), middle (24-36 hpi) and late (48-72 hpi) stages of R.solaniAG3-TB infection. These pathogenic factors included toxins, CAZymes, CWDEs, 807 potential secreted proteins and 78 cysteine-rich small proteins ( Figure 3 A in Figure 1). Based on the potential secreted proteins screened, 10 candidate pathogenic secreted proteins were identified based on RPKM>100 and Reads>200. The relative expression levels of the candidate secreted proteins were verified by real-time quantitative RT-qPCR. The results are shown in Figure 1. Figure 3As shown in B, the relative expression levels of the 10 candidate DEGs were upregulated at 12 hpi, 48 hpi, and 72 hpi. Among them, RsDN3377 and RsDN20094 were significantly upregulated at 48 h after inoculation, upregulated by 5000-fold and 100000-fold, respectively. RsDN21085, RsDN22904, RsDN22462, and RsDN22450 were upregulated to the highest level at 72 h after inoculation. The results of RT-qPCR were consistent with those of RNA-seq, indicating the accuracy of the RNA-seq results.

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

[0069] In order to obtain candidate protein gene fragments, 10 full-length fragments of candidate protein genes were designed with reference 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) were amplified according to the PCR reaction. The PCR reaction system was 20μL, including 1μL template; 0.5μL each of 10μM positive and negative primers; 10μL 2×taq Mix; 7μL ddH2O. The PCR reaction conditions are: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s; 60℃ annealing for 30s; 72℃ extension for 1min (fragment length is about 350bp, about 1kb / min), 35 cycles; 72℃ extension for 8min. The PCR product was run on 1×TAE 1% agarose gel electrophoresis to identify the target fragment. To obtain the target fragment, the target fragment was cut and recovered according to the gel recovery method.

[0070] Table 2 Specific primers for candidate genes

[0071]

[0072]

[0073] The pGR106 vector was double-digested with restriction endonucleases (Cal I and Sal I), with a total digestion system of 50 μL. The digestion products were determined 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 recovered and purified PCR target product and the pGR106 vector recovered by double enzyme digestion were recombined and ligated using T4 ligase DNA Ligation Kit (Beijing, China). The ligation system included: 1 μL vector fragment (60 ng / μL), 4 μL target fragment (20 ng / μL~50 ng / μL), 5 μL Solution I, and ligated overnight at 16°C. The ligation product was transformed into Escherichia coli competent DH5α for culture. The transformation method was as follows: Escherichia coli competent DH5α stored at -80°C was placed on ice to melt; 5 μL of the ligation product was added to 30 μL competent cells, gently mixed with a pipette, and placed on ice for 15 min; immediately heat-shocked in a 42°C water bath for 45 s, and ice-bathed for 2 min; 800 μL LB liquid culture medium (without antibiotics) was added and cultured in a shaking table at 37°C to revive the bacteria for 60 min; the bacteria were collected and plated on LB solid culture medium containing Amp resistance, and inverted cultured in a 37°C incubator for 14 h. Single colonies of E. coli grown on Kan-resistant medium were picked and shaken at 200 rpm for 7 ± 1 h in liquid LB with corresponding antibiotics, and PCR was performed on the culture solution and identified by 1% agarose gel electrophoresis. The culture solution 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 LB liquid medium for 12 h, and PCR and gel electrophoresis were performed using specific primers to determine that the band size was consistent with the expected target fragment size. The culture solution with accurate length was sent for sequencing (Sangon Biotech Co., Ltd., Shanghai), and the sequencing results were compared using DNAMAN software to extract the recombinant plasmid. 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 electric shock cup in advance and soak it in 75% alcohol for 2 hours;

[0076] 2) Rinse repeatedly with distilled water for 3 times, rinse with ultrapure water for 6 to 8 times, and soak in 2 mL of anhydrous ethanol for 30 minutes;

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

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

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

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

[0081] 7) Evenly spread the plate with rifampicin (Rif) and kanamycin (Kan) resistance, invert it in a 28°C incubator for 48 hours, and after the bacteria on the plate grow to a certain size, pick the bacteria and shake them in liquid LB containing Rif+Kan for 48 hours, and perform colony PCR and 1% agarose gel electrophoresis for identification;

[0082] 8) The strains of the same size were shaken in large quantities (20 mL). After 48 hours, when the bacterial solution turned orange-yellow as a whole, the solution was centrifuged at 4000 rpm for 10 min to recover the bacterial cells.

[0083] The cells were suspended in an infiltration buffer (preparation method: 4 mL 1 M MES; 4 mL 1 M MgCl2; 400 μL 150 mM As diluted to 400 mL sterile distilled water) and the OD was measured using a spectrophotometer. 600 The value was adjusted to within 0.5, and then placed in the dark at room temperature for 2 hours. A 1mL syringe was used to draw the bacterial suspension and injected into the back of the tobacco leaf. The injection volume for each injection site was 200μL. After injection, the symptoms were observed and photographed after 5 days of culture. The results are shown in Figure 4 .

[0084] Based on the gene expression results of 10 candidate secretory proteins screened at different inoculation times in Example 2, the full length of 10 candidate genes were successfully cloned. The mouse protein BAX, which can induce overexpression in plants to cause plant immune hypersensitivity reactions, 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 cause immune-related allergic reactions in plants, the candidate full-length cDNA was constructed and recombinant plasmids were injected into N. benthamiana leaves based on the Agrobacterium infiltration transient expression method, and the necrotic symptoms at the inoculated site were observed to identify the molecular functions of the candidate proteins. Figure 4 As shown in A, all 10 candidate genes were infiltrated into N. benthamiana leaves for 5 days, and only the protein transiently expressed by the Agrobacterium carrying the gene RsDN3377 could be observed to cause cell death symptoms. To determine the accumulation of hydrogen peroxide (H2O2) and cell death in the Agrobacterium-infiltrated leaves, the inoculation site was stained, and obvious H2O2 accumulation and cell death were observed in the stained site containing the transiently expressed RsDN3377 protein. Figure 4 As shown in Figures B and C, RsDN3377 accumulated more H2O2 and induced cell death compared with the negative GFP control.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

1.

2. The tobacco target spot pathogenic factor RsDN3377 according to claim 1, characterized in that: The nucleotide sequence of the tobacco target spot pathogenic factor RsDN3377 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 pathogenicity factor RsDN3377 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 according to claim 1.

6. The method for screening recombinant bacteria according to claim 5, characterized in that: The specific steps are as follows: (1) Obtaining gene sequences by screening pathogenic proteins secreted by R.solaniAG3-TB during infection; (2) Designing specific primer pairs for the gene sequence and amplifying to obtain the target gene fragment; (3) connecting the target fragment to the vector to obtain a connection product; (4) Transforming the ligated product into Escherichia coli and culturing it to obtain a recombinant plasmid; (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.

7. Use of the tobacco target spot pathogenic factor RsDN3377 according to claim 1 or 2, the recombinant plasmid according to claim 3 or 4, the recombinant bacterium according to claim 5, or the recombinant bacterium obtained by the screening method according to claim 6 in screening agents against tobacco target spot disease.

8. The use according to claim 7, characterized in that: 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.

9. Use of the tobacco target spot pathogenic factor RsDN3377 according to claim 1 or 2, the recombinant plasmid according to claim 3 or 4, the recombinant bacterial cell according to claim 5, or the recombinant bacterial cell obtained by the preparation method according to claim 6 in screening varieties resistant to tobacco target spot disease.

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