A plant disease-resistant gene NtWRKY50 and its application in tobacco resistance to bacterial wilt

Abstract

The present invention relates to the technical field of crop disease control, and particularly to a plant disease-resistant gene NtWRKY50 and its application in tobacco resistance to bacterial wilt. The nucleotide sequence of the plant disease-resistant gene NtWRKY50 of the present invention is shown in SEQ ID NO.1. The present invention further transfers the NtWRKY50 gene into tobacco for functional verification. The results show that NtWRKY50 is a nuclear localization protein, and its expression is induced by the infection of the plant bacterial wilt pathogen; overexpression of NtWRKY50 can enhance the resistance of tobacco to bacterial wilt, and silencing of the NtWRKY50 gene has no significant effect on plant disease resistance; NtWRKY50 can be induced by various biotic or abiotic stresses; overexpression of NtWRKY50 significantly promotes the SA level, but inhibits the pathogen-induced JA accumulation. These data indicate that overexpression of NtWRKY50 leads to changes in the contents of SA and JA, increases the expression of defense-related genes, and enhances the resistance of plants to Ralstonia solanacearum. The present invention provides a gene resource for cultivating new tobacco varieties resistant to bacterial wilt by means of genetic engineering, and has important application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of crop disease control, and particularly to a plant disease-resistant gene NtWRKY50 and its application in tobacco resistance to bacterial wilt. Background Art

[0002] Bacterial wilt is a soil-borne vascular disease caused by Ralstonia solanacearum (bacterial wilt pathogen). The bacterial wilt pathogen is one of the most destructive plant pathogens in the world and ranks second among the top ten plant pathogenic bacteria. The bacterial wilt pathogen infects more than 50 families and over 450 plant species, including solanaceous crops, bananas, peanuts, ginger, ornamental plants and forest trees. Among solanaceous crops, tomatoes, potatoes, tobacco and peppers are the main representatives, and new varieties and new hosts are constantly being reported. Due to its wide host range and geographical distribution, it causes serious economic losses (15% - 95%) worldwide, especially in developing countries in tropical regions, and is an important limiting factor in the production of many crops and cash crops. Only for potatoes, the global annual loss is 1 billion US dollars (Elphinstone, 2005#1088). The increase in temperature caused by the greenhouse effect has expanded its distribution range to higher latitudes. Due to the lack of effective eradication measures, continuous production on contaminated land and the ability of the pathogen to survive in moist soil, water pools, plant residues or asymptomatic weed hosts for many years, this disease has been difficult to control. Currently, the main methods for controlling bacterial wilt are the use of chemical pesticides and agricultural measures, but this brings many problems, such as environmental pollution and induction of pathogen resistance. Therefore, the application of disease-resistant varieties is particularly important.

[0003] The plant disease resistance response mechanism is very complex and diverse. There are a series of regulatory factors and genes during the interaction between plants and pathogens, the transduction of disease-resistant signals and the occurrence of defense responses, forming a complex regulatory network. Signal molecules can be generated through different pathways, and the same pathogen may also stimulate different disease-resistant signals. In recent years, many domestic and foreign researchers have been committed to finding genes related to disease resistance and studying disease resistance mechanisms. Various transgenic disease-resistant plants have been successively established and applied to agricultural production. The research on plant disease resistance mechanisms and the application of plant genetic engineering technology not only open up a new way for the improvement of crop varieties, but also provide new ideas for environmental protection and maintaining ecological balance, with particularly important research significance and broad application prospects.

[0004] In recent years, more and more evidence has shown that the overexpression or silencing of some WRKY transcription factors in plants can affect disease resistance. Arabidopsis thaliana, as a model crop, has the most research results on WRKY proteins. It has been confirmed that WRKY proteins are negative regulators of plant resistance, such as Arabidopsis thaliana AtWRKY4 (Lai, 2008#620), AtWRKY27 (Mukhtar, 2008#624), AtWRKY11 / 17 (Journotcatalino, 2006#1557), and AtWRKY48 (Xing, 2008#1567). Some studies have also found that Arabidopsis thaliana WRKY proteins positively regulate plant resistance, such as AtWRKY75 (Encinas-Villarejo, 2009#1569), AtWRKY3 / 4 (Lai, 2008#1555), and three partially functionally redundant WRKY genes, AtWRKY18, AtWRKY40, and AtWRKY60 (Xu, 2006#348).

[0005] In addition, multiple WRKY transcription factors have been found to regulate the plant's defense response to Ralstonia solanacearum. For example, the Arabidopsis thaliana R protein RRS1 (AtWRKY52) activates the ETI response by specifically recognizing the type III effector protein PopP2 secreted from Ralstonia solanacearum (Deslandes et al., 2002; Deslandes et al., 2003). Both Capsicum annuum CaWRKY40 and Gossypium hirsutum GhWRKY39-1 can enhance the resistance or tolerance of tobacco to Ralstonia solanacearum (Dang, 2013#1333), (Shi, 2014#350); on the contrary, GhWRKY40 (Wang, 2014#351), CaWRKY58 (Wang, 2013#361), AtWRKY53 (Hu, 2008#530), and AtWRKY27 (Mukhtar, 2008#1571) negatively regulate the plant's resistance to Ralstonia solanacearum.

[0006] In view of the above background technology, the present invention is based on previous transcriptome data. During the process of tobacco infected with Ralstonia solanacearum, a WRKY-like gene was identified to be significantly induced. Nucleotide BLAST showed that the predicted WRKY-like gene had 100% similarity with NtWRKY50, and it was named NtWRKY50. By constructing overexpression and RNAi transgenic tobacco plants and measuring the expression of resistance and defense-related genes, it is speculated that NtWRKY50 may be involved in the defense response of tobacco, providing a theoretical basis for the molecular mechanism of plant resistance to Ralstonia solanacearum. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a plant disease-resistant gene NtWRKY50 and its application in tobacco resistance to bacterial wilt, which provides a gene resource for cultivating new tobacco varieties resistant to bacterial wilt by means of genetic engineering and has important application prospects.

[0008] The present invention solves the above technical problems through the following technical means:

[0009] A plant disease-resistant gene NtWRKY50, the nucleotide sequence of the NtWRKY50 gene is shown in SEQ ID NO.1. The amino acid sequence encoded by the NtWRKY50 gene is shown in SEQ ID NO.2. The NtWRKY50 gene can significantly improve the resistance of plants to Ralstonia solanacearum.

[0010] Based on previous transcriptome data, during the process of tobacco infected with bacterial wilt, a WRKY-like gene was identified to be significantly induced. Nucleotide BLAST showed that the predicted WRKY-like gene had 100% similarity with NtWRKY50 and was named NtWRKY50. Primers were designed according to its predicted cDNA sequence, and its expression pattern was analyzed by QRT-PCR. Primers were designed according to the predicted NtWRKY50 cDNA sequence, and polymerase chain reaction was carried out to amplify this gene. The results showed that the NtWRKY50 sequence was 576 base pairs, encoding 191 amino acids, and its nucleotide sequence was shown in SEQ ID NO.1. The protein encoded by NtWRKY50 contains a WRKY domain WRKYGKK and a zinc finger motif CX4CX23HXH. Compared with the typical WRKYGQK sequence, one base G was replaced by K, and its encoded amino acid sequence was shown in SEQ ID NO.2.

[0011] In addition, the present invention also provides an overexpression vector containing the plant disease-resistant gene NtWRKY50, and a construction method of the overexpression vector: using cDNA as a template for NtWRKY50 PCR amplification, adding a Bg1II restriction enzyme site to the upstream primer and a BstEII restriction enzyme site to the downstream primer, and introducing 6 HIS proteins at the protein end. The PCR amplified NtWRKY50 fragment was recovered by electrophoresis and gel cutting, and after double digestion, it was ligated to the pVCT2024 expression vector and placed downstream of the CaMV35S promoter to obtain an overexpression vector driven by the CaMV35S promoter for the NtWRKY50 gene. Overexpression of NtWRKY50 can inhibit the growth of Ralstonia solanacearum and delay the development of tobacco wilt symptoms.

[0012] In addition, the present invention also provides an RNAi interference vector containing the plant disease-resistant gene NtWRKY50, and a construction method of the RNAi interference vector: using the pBWA(V)HS vector, PCR amplifying the forward target, reverse target and the middle loop of NtWRKY50, and then through restriction enzyme digestion and ligation reactions, inserting them downstream of the CaMV35S promoter. The RNAi interference vector of the NtWRKY50 gene does not cause a change in the resistance of tobacco plants to Ralstonia solanacearum.

[0013] Finally, the present invention also discloses the application of the plant disease-resistant gene NtWRKY50 or the overexpression vector in the genetic engineering of tobacco resistant to Ralstonia solanacearum. The overexpression vector is constructed by placing NtWRKY50 downstream of the CaMV35S promoter, and through Agrobacterium-mediated transformation, transgenic tobacco plants overexpressing NtWRKY50 are obtained, and their resistance is identified. The results prove that the NtWRKY50 gene can positively regulate the resistance of tobacco to Ralstonia solanacearum.

[0014] The plant disease-resistant gene NtWRKY50 of the present invention can positively regulate the resistance of tobacco to Ralstonia solanacearum, and interfering with the expression of NtWRKY50 does not change the disease resistance of tobacco. In addition, NtWRKY50 positively regulates the hormone-mediated defense pathway, especially after being infected by pathogens. In addition, NtWRKY50 responds to various hormone or stress inductions, and the induction by hormones is better. The present invention provides a gene resource for cultivating new tobacco varieties resistant to Ralstonia solanacearum by genetic engineering means, and has important application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the map of the expression vector pVCT2040 in the construction of the overexpression vector of the NtWRKY50 gene of the present invention;

[0016] Figure 2 is the map of the RNAi interference vector containing the NtWRKY50 gene of the present invention;

[0017] Figure 3 is the expression situation of the NtWRKY50 gene of the present invention during the infection of Ralstonia solanacearum. Among them, 3A is the symptom of the Yunyan 87 tobacco plant inoculated with Ralstonia solanacearum, 3B is the growth situation diagram of Ralstonia solanacearum in the tobacco roots, and 3C is the relative expression quantity diagram of NtWRKY50 in Yunyan 87 after inoculating Ralstonia solanacearum;

[0018] Figure 4 is the comparison diagram of the WRKY domain WRKYGKK and the zinc finger motif contained in the protein encoded by NtWRKY50 of the present invention with the typical WRKYGQK sequence;

[0019] Figure 5 is the grouping diagram of the WRKY transcription factors of the present invention;

[0020] Figure 6 This is the subcellular localization result diagram of NtWRKY50 of the present invention;

[0021] Figure 7 This is the diagram showing the influence of overexpression of NtWRKY50 of the present invention on the resistance to Ralstonia solanacearum. Among them, 7A is the diagram showing the expression levels of NtWRKY50 in different overexpression transgenic plant lines detected by QRT-PCR, 7B is the statistical chart of the incidence rates of NtWRKY50-OE2 and WT plants inoculated with Ralstonia solanacearum, and 7C is the statistical chart of the growth conditions of Ralstonia solanacearum in the roots of NtWRKY50-OE2 and WT;

[0022] Figure 8 This is the histochemical staining diagram of tobacco infected by Ralstonia solanacearum of the present invention. Among them, 8A is 3,3'-diaminobenzidine staining, and 8B is nitroblue tetrazolium staining;

[0023] Figure 9 This is the diagram showing the influence of the RNAi interference vector of NtWRKY50 of the present invention on the resistance to Ralstonia solanacearum. Among them, 9A is the diagram showing the expression levels of NtWRKY50 in different RNAi transgenic plant lines detected by QRT-PCR, 9B is the statistical chart of the incidence rates of NtWRKY50-OE2 and WT plants inoculated with Ralstonia solanacearum, and 9C is the statistical chart of the growth conditions of Ralstonia solanacearum in the roots of NtWRKY50-OE2 and WT;

[0024] Figure 10 This is the bar chart of QRT-PCR analysis of defense-related genes before and after inoculation with Ralstonia solanacearum of the present invention; among them, 10A is the QRT-PCR analysis diagram before inoculation, and 10B is the QRT-PCR analysis diagram 1 day after inoculation;

[0025] Figure 11 This is the bar chart of the endogenous free SA and JA contents of the present invention. Detailed implementation manners

[0026] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0027] The nucleotide sequence of a plant disease-resistant gene NtWRKY50 of the present invention is shown in SEQ ID NO.1. The amino acid sequence encoded by the NtWRKY50 gene is shown in SEQ ID NO.2.

[0028] The culture media used in the present invention are as follows:

[0029] TTC1L: 1 g of casein hydrolysate, 10 g of peptone, 10 g of glucose, 20 g of agar, pH = 7.0. The culture medium is melted and cooled to 50 °C before use, and then 0.05 g of TTC (2,3,5-triphenyltetrazolium chloride) is added;

[0030] NB1L: 3 g of beef extract, 1 g of yeast powder, 5 g of peptone, 10 g of glucose, pH = 7.0;

[0031] YEB1L: 5 g of beef extract, 1 g of yeast extract, 5 g of peptone, 5 g of sucrose, 0.5 g of MgSO4·H2O, pH = 7.0;

[0032] LB1L: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar;

[0033] SMSA1L: 10 g of peptone, 5 mL of glycerol, 1 g of casein hydrolysate, 1000 mL of deionized water, pH = 7.0 - 7.2, 15 g of agar powder, sterilized at 121 °C for 20 minutes. The medium is melted and cooled to 50 °C before use and the following substances are added: 100 mg / L of polymyxin B, 5 mg / L of crystal violet, 50 mg / L of triphenyltetrazolium chloride, 25 mg / L of bacitracin, 0.5 mg / L of penicillin, 5 mg / L of chloramphenicol, 100 mg / L of cycloheximide.

[0034] Example 1: Preparation of sterile tobacco seedlings

[0035] In this example, wild-type tobacco Yunyan seeds from Chongqing Tobacco Company were used. They were soaked in an alcohol solution with a mass concentration of 75% for 1 minute, taken out and soaked in sodium hypochlorite with a mass concentration of 10% for 10 minutes, then taken out and washed 5 times with sterilized water, and sown on the MS solid medium provided by Qingdao Haibo Biotechnology Co., Ltd., and placed in an artificial climate chamber at 25 - 28 °C, and cultured and grown in a climate chamber with a light cycle of 16 h light / 8 h darkness.

[0036] Example 2: Construction of NtWRKY50 gene overexpression vector and RNAi interference vector

[0037] Fluorescence real-time quantitative PCR: RNA was extracted using the plant total RNA extraction kit provided by Tiangen Biochemical Technology Co., Ltd., cDNA was synthesized using the BIO-RAD cDNA Synthesis Kit, and fluorescence quantitative PCR (QRT-PCR) was performed using BIO-RAD, C1000Touch TM Thermal Cycler, SsoFAST TM Eva Green Supermix, and the detection system was CFX96 real-time system. The PCR amplification reaction system was 20 μl, containing 10 μl of SYBR mix, 1 μl of cDNA, 1 μl of forward primer, 1 μl of reverse primer and 7 μl of H2O. Reaction conditions: 95 °C for 3 min, 40 cycles: 95 °C for 10 s, 55 °C for 30 s. Each PCR reaction was carried out in three biological replicates and passed through 2-ΔΔCt The method calculates gene expression. The reference genes are UBI3 and NtEF1α.

[0038] In the construction of the overexpression vector in this example, the expression vector used is the pVCT2024 vector from Teacher Zhang Xingguo of the College of Horticulture, Southwest University. Its map is as Figure 1 shown. Using cDNA as a template for NtWRKY50 PCR amplification, the BglII restriction enzyme site was added to the upstream primer, the BstEII restriction enzyme site was added to the downstream primer, and 6 HIS proteins were added to the protein terminal primer. After electrophoresis and gel cutting, the NtWRKY50 fragment amplified by PCR was recovered, double digested and then ligated to the large plasmid fragment of the pVCT2024 expression vector, and inserted downstream of the CaMV35S promoter to obtain an overexpression vector driven by the CaMV35S promoter for the NtWRKY50 gene. Transformed into Escherichia coli DH5α, plated on LB+Kan, and positive clones were verified by colony PCR and sent to BGI for sequencing. The correct clones were selected to extract plasmids (Tiangen plasmid miniprep kit) and transformed into Agrobacterium tumefaciens EHA105, and positive clones were verified by colony PCR. Cultured in liquid YEB+Kan for 48 h, 0.2 mL was aspirated and an equal volume of 50% sterilized glycerol was added and stored at -80 °C.

[0039] Construction of the RNAi interference vector for the NtWRKY50 gene: Using the pBWA(V)HS vector, first PCR amplify the forward target, reverse target and the middle loop of NtWRKY50, and then carry out a restriction enzyme digestion and ligation reaction at 37 °C for 2 h and insert it downstream of the CaMV35S promoter. The following is the enzyme digestion and ligation reaction system for every 10 μl: loop 1.5 μl, forward and reverse targets each 1.5 μl, empty vector 1.5 μl, Eco31I 0.5 μl, T4 ligase 0.5 μl, T4 buffer 1 μl, and ddH2O was added to make up to 10 μl. The map of the RNAi interference vector for the NtWRKY50 gene is as Figure 2 shown.

[0040] Example 3: Culturing transgenic tobacco plants

[0041] Step 1: Streak the EHA105 bacterial solution containing the constructed plasmid of the NtWRKY50 gene on a YEB+Kan plate and culture it at 20 °C for 48 h. Pick a single colony into 5 mL of YEB medium and culture it at 280 rpm and 28 °C for 48 h. Expand the culture 50 mL overnight at a ratio of 1:100 to 1:50 until the optical density OD = 0.6, then centrifuge to collect the bacteria, and suspend it with 50 mL of MS liquid medium provided by Qingdao Haibo Biotechnology Co., Ltd. to obtain an infection bacterial solution for standby.

[0042] Step 2: Transfer about 10 mL of the prepared Agrobacterium liquid into an empty sterile Petri dish. Remove the midrib and leaf margin of tobacco leaves and cut them into uniform small pieces of 0.5 - 0.8 cm × 0.5 - 0.8 cm. Transfer the leaves into the infection liquid, gently shake and infect for 5 - 8 min. Use forceps to clamp the leaf discs onto sterilized filter paper to absorb the residual liquid on the leaves. Transfer the small leaves onto the co-culture medium MS, attach a layer of sterilized filter paper on the surface of the medium, place the leaves face up on the filter paper, seal the dish with sealing film as the experimental group. At the same time, place the leaf discs not infected with Agrobacterium on the same co-culture medium as the negative control, denoted as the control group and represented by WT.

[0043] Step 3: Place both the experimental group and the control group in step 2 in the dark and co-culture at a temperature of 28 °C for 2 - 3 days. After co-culture, immerse the transformed leaf discs in MS liquid medium for 3 - 5 min. Absorb the residual liquid with sterile filter paper and transfer them to the selection and differentiation medium (MS + KT + NAA + Kan + Carb). At the same time, perform the same treatment on the control leaf discs. Then culture in a light incubator under the conditions of 24 °C and 16 h of light.

[0044] Step 4: Regeneration of roots after transformation. Approximately 6 weeks after transformation, cut the differentiated resistant buds and transfer them onto the selection rooting medium (MS + Kan + Carb). Roots can be seen after 10 - 15 days. After PCR and QRT-PCR detection, perform a large amount of asexual propagation on the NtWRKY50 overexpression and RNAi transgenic sterile seedlings, and transfer them to peat substrate for potted cultivation after rooting.

[0045] Example 4: Method for inoculating transgenic tobacco plants

[0046] Leaf injection inoculation: Ralstonia solanacearum CQPS-1 was isolated from Pengshui area, Chongqing in this laboratory and stored at -80 °C. Streak-culture Ralstonia solanacearum CQPS-1 on NA medium for 48 h, select monoclonal colonies and culture overnight in NB until OD 600 <1, dilute the bacterial liquid to OD = 0.01, remove the needle of the syringe and inject 20 μl of the bacterial liquid into the leaves of tobacco plants at 8 weeks old from the back; in addition, inject an equal amount of sterilized ddH2O into the control group.

[0047] Root irrigation inoculation: Activate Ralstonia solanacearum CQPS-1 on NA medium, inoculate it in NB medium and shake the bacteria overnight (OD 600 <1), dilute it with sterilized ddH2O to OD 600 = 0.1 (10 8(CFU / mL) was used for inoculation. Tobacco plants at 8 weeks old were inoculated with 10 mL of Ralstonia solanacearum solution around the base of the stem; in addition, the control group was inoculated with an equal amount of sterilized ddH2O. The experiment was repeated three times, and the SPASS software version 13.0 (t-test, P < 0.05) was used to analyze whether the differences were significant.

[0048] The disease severity was classified as follows: grade 0, the whole plant was disease-free; grade 1, there were occasional chlorotic spots on the stem or less than half of the leaves on the diseased side or slight wilting at the top; grade 2, there were black streaks on the stem, but it did not reach the top of the plant or more than half of the leaves on the diseased side or the leaves above the waist wilted; grade 3, the black spots on the stem reached the top of the plant or all the leaves wilted or most of the leaves wilted; grade 4, the diseased plants died completely.

[0049] Root-injuring inoculation: The steps of root-injuring inoculation were basically the same as those of root irrigation inoculation, except that before the inoculation solution was poured in, the roots were vertically scratched on both the left and right sides at a position 1 cm away from the base of the stem with a small knife. At 30 min, 1 d, 3 d, and 5 d after inoculation, the roots and stems of the plants were collected, ground in a mortar, diluted into different gradients, and spread on NA medium, and the bacterial content was counted and detected after 48 h.

[0050] Example 5: Identification of NtWRKY50 gene and cDNA sequence

[0051] Based on the previous transcriptome data of the laboratory, during the process of tobacco infected with Ralstonia solanacearum, a WRKY-like gene was significantly induced. Nucleotide BLAST showed that the predicted WRKY-like gene had 100% similarity with NtWRKY50, so it was named NtWRKY50. The expression of NtWRKY50 gradually increased with the development of the disease process, and it was upregulated by more than 70 times on the third day. Primers were designed according to its predicted cDNA sequence, and its expression pattern was analyzed by QRT-PCR. To verify the transcriptional pattern of the NtWRKY50 gene under the infection of Ralstonia solanacearum, Yunyan 87 WT plants at 8 weeks old were inoculated with 10 8 CFU / m of bacteria at a concentration of 10 mL. One day after inoculation, no wilt symptoms were observed, but a small amount of bacteria was detected in the roots. As the disease process developed, the bacterial concentration increased significantly, and severe wilt symptoms appeared on the 3rd day after inoculation, as Figure 3 A and Figure 3 B showed. In addition, the roots and the base of the stem of the plants were collected at 0, 1, 3, and 5 days respectively, total RNA was extracted, and cDNA was further generated by reverse transcription. QRT-PCR analysis showed that the NtWRKY50 gene began to be induced and expressed at 1 d, and the expression level reached the peak at 5 d with the increase of time, as Figure 3 C showed. The above results indicate that the NtWRKY50 gene was continuously induced and activated during the infection of Ralstonia solanacearum.

[0052] Primers were designed according to the predicted NtWRKY50 cDNA sequence, and polymerase chain reaction was carried out to amplify this gene. The results showed that the NtWRKY50 sequence was 576 base pairs and encoded 191 amino acids. The protein encoded by NtWRKY50 contained a complete WRKY domain WRKYGKK and a zinc finger motif (CX4CX 23 HXH). Compared with the typical WRKYGQK sequence, one base G was replaced by K, as Figure 4 shown. The nucleotide sequence of NtWRKY50 is as shown in SEQ ID NO.1, and the amino acid sequence encoded by the NtWRKY50 gene is as shown in SEQ ID NO.2.

[0053] WRKY transcription factors are generally divided into three major categories: WRKYI, WRKYII, and WRKYIII. The WRKYII category is further divided into five subgroups, namely IIa, IIb, IIc, IId, and IIe. Phylogenetic analysis of NtWRKY50 was performed using MEGA3.1 and the neighbor-joining method. The results showed that NtWRKY50 was classified into the IIc subgroup together with Arabidopsis thaliana AtWRKY8 and AtWRKY28, as Figure 5 shown. This subgroup contains a large number of variations in the WRKYGQK heptapeptide sequence, which is consistent with the results Figure 4 indicated.

[0054] Example 6: Effect of overexpression of NtWRKY50 on resistance to bacterial wilt

[0055] Subcellular localization of NtWRKY50: The open reading frame sequence of NtWRKY50 was constructed into the pEGAD vector, controlled by the cauliflower mosaic virus CaMV35S promoter, and fused to the C-terminus of the green fluorescent protein (GFP) gene. Transient expression induced by Agrobacterium was used in tobacco leaves, and the results showed that NtWRKY50 fused with GFP was located in the nucleus of leaf cells, as Figure 6 shown.

[0056] An overexpression vector was constructed, with NtWRKY50 placed downstream of the CaMV35S promoter, and transgenic tobacco plants overexpressing NtWRKY50 were obtained through Agrobacterium-mediated transformation. Among 7 independent transgenic lines, the NtWRKY50 expression level of the NtWRKY50-OE2 line was the highest, as Figure 7 shown in A. Tobacco plants with an 8-week-old tobacco age were inoculated by root irrigation with 10 mL of Ralstonia solanacearum at a concentration of 10 8 CFU / mL. The results were as Figure 7 shown in B, and the incidence of NtWRKY50-OE2 plants was lower than that of WT plants. The evaluation of the bacterial content in the roots was carried out by the method of inoculating wounded roots, and the results were as Figure 7As shown in C. The results showed that the growth of Ralstonia solanacearum was inhibited in NtWRKY50-OE2, significantly lower than that in WT plants.

[0057] In addition, for histochemical staining, after inoculating Ralstonia solanacearum into WT plants and NtWRKY50-OE2 plants by leaf injection for 1 day, they were stained with 1 mg / mL 3,3'-diaminobenzidine or 0.1 mg / mL nitroblue tetrazolium for 24 h and 6 h respectively in the dark environment at 25 °C. The leaves stained with 3,3'-diaminobenzidine were boiled in a mixed solution of lactic acid: glycerol: absolute ethanol with a volume ratio of 1:1:3, and then decolorized in absolute ethanol overnight. The leaves stained with nitroblue tetrazolium were directly decolorized in absolute ethanol overnight. Then the leaves were soaked in 100% ethanol, stored at room temperature and imaged. The results were as Figure 8 shown. The results showed that the content of ROS was higher in NtWRKY50-OE2 as shown by 3,3'-diaminobenzidine and nitroblue tetrazolium staining. Therefore, these results indicated that NtWRKY50 could positively regulate the resistance of tobacco to Ralstonia solanacearum.

[0058] To further determine the role of NtWRKY50 in regulating plant disease resistance, we used the antisense silencing technique RNAi to interfere with the expression of NtWRKY50 in tobacco. The transcriptional levels of NtWRKY50 in 7 RNAi lines S1-S7 were detected by QRT-PCR, as Figure 9 shown in A. The results showed that the transcriptional level of NtWRKY50 was the lowest in line S2. Similar to the NtWRKY50 overexpressing plants, we inoculated them with Ralstonia solanacearum and detected their resistance. The results showed that there were no obvious differences in the disease symptoms and bacterial growth between the NtWRKY50-silenced line S2 and the WT line, as Figure 9 shown in B and 9C. These results indicated that interfering with the expression of NtWRKY50 did not change the disease resistance of tobacco, and NtWRKY50 was functionally redundant with other transcription factors.

[0059] Example 7: Induction of NtWRKY50 by Abiotic Stress

[0060] To study the responses of NtWRKY50 to various abiotic stresses, the following abiotic stress treatments were carried out in this example: The leaves of tobacco seedlings with an 8-week tobacco age were sprayed with solutions of 2 mM salicylic acid (SA), 0.1 mM jasmonic acid (JA), 7 mM ethylene (ETH), 100 μM abscisic acid (ABA), 0.6 mM gibberellic acid (GA), 10 mM hydrogen peroxide (H2O2), and 200 mM sodium chloride (NaCl). The potted tobacco seedlings were placed in an environment of 38 °C for heat treatment, 15 °C for cold treatment, and subjected to wounding treatment. The plant leaves were scratched parallelly with a blade to make 8 wounds, and then RNA was extracted at different time points. The expression level of NtWRKY50 was detected by QRT-PCR, and SA was quantitatively analyzed by high performance liquid chromatography-mass spectrometry. The results showed that when WT Yunyan 87 was induced by various hormones, the expression of NtWRKY50 increased, especially with the best effect of SA, reaching the highest value at 24 h, which was 10 times higher than that before induction; ETH and JA treatments could induce NtWRKY50 to increase by 5 or 7 times; other stress treatments also induced the up-regulation of NtWRKY50; cold, heat, and NaCl treatments had a greater impact on NtWRKY50, and the peak value could reach 4-6 times that before induction; the induction effects of H2O2 and wounding treatment were the least obvious, and the highest value was only 2 times that of the control. In summary, NtWRKY50 responds to various hormone or stress inductions, and the induction effect of hormones is better.

[0061] Example VIII: Analysis of Plant Hormones

[0062] SA, JA, and ET are important defense signal molecules. Next, the effects of overexpression of NtWRKY50 on the expression of SA-, JA-, and ET-responsive genes will be analyzed.

[0063] By inoculating the roots of NtWRKY50-OE2 and WT plants with wounds, total RNA was extracted and cDNA was generated. The PR genes responsive to SA and JA / ET, such as PR1A / C, PR1B, PR2, PR3, the genes related to ET biosynthesis and signal markers, such as ACC Oxidase, ACS1, and EFE26, the HR-related genes HSR201 and H1N1, and the ROS scavenging enzyme genes, including catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST) were analyzed by QRT-PCR. Before the infection of Ralstonia solanacearum, compared with WT, the gene expressions of PR1B, PR2, ACS1, and EFE26 in NtWRKY50-OE2 were up-regulated by 3-6 times, while PR3 and H1N1 increased by 20-30 times, as Figure 10 shown in A, and there were no obvious changes in other genes. However, after inoculation, the transcriptional levels of most genes in NtWRKY50-OE2 were up-regulated by 50-800 times compared with WT, as Figure 10As shown in B. However, ROS scavenging enzymes showed different regulatory patterns: the expression level of CAT was lower in NtWRKY50-OE2 than in WT, while the expression level of GST was 10 times that of WT. These results indicate that NtWRKY50 positively regulates the hormone-mediated defense pathway, especially after pathogen infection.

[0064] Plant hormones, especially SA and JA, play very important roles in resisting disease infection. To investigate whether the improved resistance phenotype of NtWRKY50-OE2 is related to changes in endogenous hormone levels, we detected the levels of SA and JA in NtWRKY50-OE2 and WT plants by GC-MS before and after inoculation with Ralstonia solanacearum. As Figure 11 shown, before inoculation, a small amount of SA = 3.98 ng / g was detected in WT plants, while the SA content in NtWRKY50-OE2 was 51.59 ng / g, about 13 times that of WT plants. However, after inoculation with Ralstonia solanacearum, the SA level increased sharply to 65.81 ng / g in WT plants, but only slightly increased in NtWRKY50-OE2, and the two contents were comparable. In contrast, the JA contents of WT and NtWRKY50-OE2 were similar before inoculation, 3.27 ng / g and 3.30 ng / g respectively. After inoculation, the JA level in WT increased significantly to 32.34 ng / g, about 10-fold increase, but hardly changed in NtWRKY50-OE2. These results indicate that NtWRKY50 has different effects on the contents of SA and JA. Overexpression of NtWRKY50 specifically promotes SA accumulation, but inhibits JA expression after pathogen infection.

[0065] The above results indicate that in the absence of inoculation, the SA and JA / ET-responsive PR genes and the genes related to ET synthesis and signal pathways in NtWRKY50-OE2 were upregulated by 3-6 or 20-30 times respectively, while the expression levels of these genes increased by 50-800 times in NtWRKY50-OE2 compared with WT after inoculation with Ralstonia solanacearum. This shows that NtWRKY50 can coordinately activate the SA and JA / ET defense pathways.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies. Sequence Listing <110> Southwest University <120> A Plant Disease Resistance Gene NtWRKY50 and Its Application in Tobacco Resistance to Ralstonia solanacearum <130> 2017 <141> 2017-09-26 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 650 <212> DNA <213> Nicotiana tabacum <400> 1 gaaagaagtc ttgaaaagtc atctcgtcaa gtccttcaaa cattttcata gcatgtcctc 60 tcctttactt tcaacaatgg aaaattttca ttactcaaac cctaatccta accctaatta 120 tggagctact gattttattg agacgccgga atttgaactc tccgattatc tctttcctgt 180 cgacgaattg agtgatgatt ttttgttaca aaatgaaatg acgtctgaat ttgttcaaag 240 tatttccagc agtggatcat attccaatcc cactccaagt tcagctaata acataaaatg 300 tacaaaaggt gtaaagaagt acaacaaggt ggatgcaaag tctagggttg catttagatt 360 taaatcggac ttggatgttt tggatgatgg atttaaatgg aggaagtatg gcaagaagat 420 ggtcaagaat cgtccaaatc caaggaatta ctacaaatgc tcaagtggag gatgcaatgt 480 gaagaaaaga gttgaaaggg acaatgagga ctcaagctat gtcatcacta cctatgaagg 540 aattcacaat caccagagcc cttctcatgt gcttcactac acacaattcc ctcccaaaaa 600 tattggcctt cataaccttc gcctttagat tttttttttt tttttttttt 650 <210> 2 <211> 191 <212> PRT <213> Nicotiana tabacum <400> 2 Met Ser Ser Pro Leu Leu Ser Thr Met Glu Asn Phe His Tyr Ser Asn 1 5 10 15 Pro Asn Pro Asn Pro Asn Tyr Gly Ala Thr Asp Phe Ile Glu Thr Pro 20 25 30 Glu Phe Glu Leu Ser Asp Tyr Leu Phe Pro Val Asp Glu Leu Ser Asp 35 40 45 Asp Phe Leu Leu Gln Asn Glu Met Thr Ser Glu Phe Val Gln Ser Ile 50 55 60 Ser Ser Ser Gly Ser Tyr Ser Asn Pro Thr Pro Ser Ser Ala Asn Asn 65 70 75 80 Ile Lys Cys Thr Lys Gly Val Lys Lys Tyr Asn Lys Val Asp Ala Lys 85 90 95 Ser Arg Val Ala Phe Arg Phe Lys Ser Asp Leu Asp Val Leu Asp Asp 100 105 110 Gly Phe Lys Trp Arg Lys Tyr Gly Lys Lys Met Val Lys Asn Arg Pro 115 120 125 Asn Pro Arg Asn Tyr Tyr Lys Cys Ser Ser Gly Gly Cys Asn Val Lys 130 135 140 Lys Arg Val Glu Arg Asp Asn Glu Asp Ser Ser Tyr Val Ile Thr Thr 145 150 155 160 Tyr Glu Gly Ile His Asn His Gln Ser Pro Ser His Val Leu His Tyr 165 170 175 Thr Gln Phe Pro Pro Lys Asn Ile Gly Leu His Asn Leu Arg Leu 180 185 190

Claims

1. Application of an overexpression vector of plant disease-resistant gene NtWRKY50 in improving the resistance of tobacco to bacterial wilt, characterized in that, Overexpression of the NtWRKY50 gene increases the expression levels of tobacco PR1B, PR2, PR3, ACS1, EFE26, and H1N1 genes, promotes SA accumulation in tobacco, and inhibits the increase in JA content in tobacco after infection with Ralstonia solanacearum; the nucleotide sequence of the NtWRKY50 gene is as shown in SEQ ID NO.1.

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