CN113229143B

CN113229143B - Application of kinase inhibitor Staurosporine in improving bacterial wilt resistance of plants

Info

Publication number: CN113229143B
Application number: CN202110131374.7A
Authority: CN
Inventors: 于峰; 刘红斌; 汪龙; 伍斗生; 李晓旭
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2021-01-30
Filing date: 2021-01-30
Publication date: 2023-06-09
Anticipated expiration: 2041-01-30
Also published as: CN113229143A

Abstract

The invention belongs to the technical field of plant disease control, and particularly relates to application of a kinase inhibitor Staurosporine in improving resistance of plant bacterial wilt. Digging FER gene related to bacterial wilt resistance, screening kinase inhibitor of FER in model plant Arabidopsis thaliana, firstly verifying resistance regulation action of kinase inhibitor on bacterial wilt in Arabidopsis thaliana, and finally verifying function of related kinase inhibitor in tobacco with serious bacterial wilt hazard, and finding that kinase inhibitor Staurosporine can inhibit kinase activity of FER, so as to raise resistance of tobacco to bacterial wilt.

Description

Application of kinase inhibitor Staurosporine in improving bacterial wilt resistance of plants

Technical Field

The invention belongs to the technical field of plant disease control, and particularly relates to application of a kinase inhibitor Staurosporine in improving resistance of plant bacterial wilt.

Background

FERRONIA (FER) is a class of receptor protein kinase conserved in the plant kingdom, and is found in plants to have the function of responding to biotic and abiotic stresses, including cold and heat stresses, salt stresses, pathogenic bacterial stresses, and the like. In particular, in recent years, it has been found that the ligand molecule RALF23 can inhibit the plant immune system through FER binding, and in the root of plants, the root-knot nematode competes with FER for binding through secretion of RALF analogues, thereby achieving the effect of inhibiting the plant immune system, so that FER plays an important role in plant immune regulation and mainly plays a role of inhibiting immunity. FER acts as a class of receptor protein kinases, and functions primarily through autophosphorylation of the kinase domain, thereby affecting kinase activity, thereby recruiting downstream proteins to complete signal transmission and functional output, and thus the kinase activity of FER plays an important role in its regulated immune response.

The kinase inhibitor Staurosporine is a potent, non-selective protein kinase inhibitor, IC that inhibits PKC, PKA, c-Fgr, and Phosphorylase kinase ₅₀ 6nM,15nM,2nM,3nM, respectively; the kinase inhibitor Staurosporine is an apoptosis inducer.

Bacterial wilt is an earth-borne bacterial disease caused by infection of pseudomonas (burkholderia) bacterial wilt (Ralstonia) and Ralstonia (Ralstonia solanacearum), and has wide host range, and can infect hundreds of plants such as tobacco, tomatoes, potatoes, peppers and the like. Tobacco bacterial wilt mainly occurs in tropical and subtropical areas, and is a large destructive soil-borne disease in tobacco production. The tobacco bacterial wilt still has various problems in the aspects of chemical and biological control and the like, and bacterial wilt has more variation types and more complex infection sources, so that a broad-spectrum bacterial wilt regulating gene in plants is searched, and the resistance of the bacterial wilt is regulated by chemical means to be a more effective strategy for controlling the bacterial wilt.

Disclosure of Invention

The invention mainly solves the technical problems that: overcomes the problems of chemical and biological prevention and control of plant bacterial wilt and provides a new prevention and control method.

The whole idea of the invention is as follows: digging FER gene related to bacterial wilt resistance, screening kinase inhibitor of FER in model plant Arabidopsis thaliana, firstly verifying resistance regulation action of kinase inhibitor on bacterial wilt in Arabidopsis thaliana, and finally verifying function of related kinase inhibitor in tobacco with serious bacterial wilt hazard, and finding that kinase inhibitor Staurosporine can inhibit kinase activity of FER, so as to raise resistance of tobacco to bacterial wilt.

The technical scheme of the invention is as follows:

the FER gene is found to be involved in the resistance process of bacterial wilt through early screening and plays a role in negative regulation.

Screening more than 1400 kinase activity inhibitors finds that THE kinase inhibitor Staurosporine can inhibit THE kinase activity of FER with high efficiency, and simultaneously detects THE influence of THE kinase inhibitor Staurosporine on THE kinase activity of HERK1, ANJEA and THE1 with strong autophosphorylation capability in FER homologous proteins, and finds that THE specificity of THE inhibitor is relatively high.

Use of a kinase inhibitor Staurosporine for increasing bacterial wilt resistance in plants.

Preferably, the kinase inhibitor Staurosporine is Staurosporine as an active ingredient.

Preferably, the concentration of staurosporine is between 10nM and 10. Mu.M.

Preferably, the concentration of staurosporine is 5 μm.

Preferably, the plant is a plant susceptible to bacterial wilt.

Preferably, the plant susceptible to bacterial wilt is tobacco, tomato or capsicum.

A plant bacterial wilt inhibitor comprising the kinase inhibitor Staurosporine.

According to the invention, through screening kinase inhibitors of FER in model plant Arabidopsis thaliana, firstly, the resistance regulation effect of the kinase inhibitors on bacterial wilt is verified in Arabidopsis thaliana, and finally, the functions of related kinase inhibitors are verified in tobacco with serious bacterial wilt, and the kinase inhibitor Staurosporine is found to inhibit the kinase activity of FER, so that the resistance of tobacco on bacterial wilt is improved.

Drawings

FIG. 1 shows the occurrence of wild type WT and FER function-deleted mutant FER-4 of Arabidopsis thaliana after bacterial infection.

FIG. 2 is a graph showing the results of screening for a chemical inhibitor of FER kinase activity.

FIG. 3 is a graph showing the result of the bacterial wilt disease development under different treatments of Arabidopsis thaliana.

FIG. 4 is a bacterial wilt resistance phenotype map of the control group, the Staurosporine treated group.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

Shaking bacterial wilt CQPS strain (strain is from document Genome sequencing of Ralstonia solanacearum CQPS-1,a phylotype I strain collected from a highland area with continuous cropping of tobacco) overnight at 28deg.C, and concentrating the bacterial liquid OD ₆₀₀ Wild-type WT and FER loss-of-function mutant from arabidopsis thaliana, adjusted to 0.1 and inoculated to 4 weeks of growthThe roots of fer-4 were irrigated with 30mL of bacterial wilt bacteria solution per plant to inoculate an equivalent dd H ₂ O plants served as a blank and were grown in long-day light at 28 ℃. The water is poured once every day, the disease development of the bacterial wilt of the plants is observed and recorded, and the disease condition of the bacterial wilt of the arabidopsis wild type WT appears when the wild type WT migrates along with time in the case of inoculating 6-15d plants, and the FER function deletion mutant FER-4 grows normally without the disease condition of the bacterial wilt.

Example 2

Based on the prokaryotic expression protein technology, a prokaryotic expression vector of the FER gene is constructed, and FER intracellular domain protein is obtained by an in vitro purification mode, and the specific operation steps are as follows:

and (3) PCR cloning: PCR amplification of FER gene fragment. PCR reaction system (50. Mu.L see Table 1),

TABLE 1 PCR reaction System Components

PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s,34 cycles, extension at 72℃for 10min, and preservation at 4℃after completion. And (3) PCR product recovery: and (3) performing gel recovery on the PCR product of the FER, and preserving at 4 ℃. The primer for PCR amplification of FER is His-FER-F/His-FER-R, and the primer sequence is as follows:

His-FER-F

5'-CCACAGCCAGGATCCGGGTGCTTACCGCAGACGTA-3' (BamHI cleavage site underlined)

His-FER-R

5'-TTTACCAGACTCGAGCTAACGTCCCTTTGGATTCATGAT-3' (underlined as Xho I cleavage site)

Prokaryotic expression vector construction: pRSF-Duet vector is digested with BamH I and Xho I, vector fragment is recovered, recovered PCR product is premixed with vector fragment, connection is carried out by utilizing homologous recombination method, competent Top10 is transformed, LB plate (containing 50mg/L Kan) is coated, single colony is obtained after overnight culture at 37 ℃, positive transformant is obtained after colony PCR identification, plasmid is extracted and sequenced after shaking overnight at 37 ℃. The resulting positive plasmid pRSF-Duet-FER and the previously constructed plasmid pGEX-4T-1-ABI2 were co-transferred into BL21 competent cells. Performing IPTG induction on BL21 strain carrying prokaryotic expression recombinant plasmid, wherein the temperature is 16 ℃ and 110rpm,16h,0.5mM IPTG; after induction, the thalli are crushed by ultrasonic, and are adsorbed and purified by Ni column, and the protein is stored at-80 ℃ after ultrafiltration.

Example 3

Screening of FER kinase Activity chemical inhibitors: prokaryotic expression and purification after ultrafiltration to give FER intracellular domain protein, kinase-Lumi was used ^TM The chemiluminescent kinase activity detection kit (Biyun, china) is used for detecting FER autophosphorylation activity by referring to the description. A kinase system containing 0.5. Mu.M FER (50 mM HEPES, 10mM MgCl2, 10mM MnCl2, 1mM EGTA, pH=7.48) was treated with an inhibitor (kinase inhibitor library from MedChemExpress, shanghai) at a final concentration of 5. Mu.M at room temperature for 30min, with the inhibitor solvent DMSO (0.05%) as a negative control, followed by addition of ATP at a final concentration of 0.25. Mu.M for 10min, followed by addition of an ATP-dependent luciferase-containing detection reagent in an equivalent volume to the reaction system for 10min and chemiluminescent assay by a multifunctional microplate reader. The kinase activity inhibitory effect was indicated by using the amount of ATP (μM) consumed in the reaction system as an index. With 65% inhibition rate as the minimum standard, 33 small molecule compounds obtained by screening 1494 animal kinase small molecule inhibitors can efficiently inhibit the autophosphorylation activity of FER, and the average inhibition effect reaches 80% (figure 2A). THE specificity of 33 inhibitors was examined using THE intracellular domains of HERK1, ANJEA and THE1, which were more autophosphorylated in THE FER homologous protein (FIGS. 2B-D), and inhibitor No. 17 kinase inhibitor was found to inhibit THE kinase activities of FER, HERK1, ANJEA and THE1 simultaneously. It was further demonstrated that the kinase inhibitor Staurosporine is an effective, non-selective protein kinase inhibitor, and thus Staurosporine has a broad-spectrum effect of inhibiting kinase activity, and thus Staurosporine was also intended to be used for verifying its function in resisting bacterial wilt, and its effect was analyzed.

Example 4

One week old Arabidopsis thaliana was placed in 1/2MS liquid medium containing 5. Mu.M kinase inhibitor StaurosporineAfter 1d of medium culture, the culture medium is divided into an experimental group and a control group; the experimental group was added with inhibitor solvent DMSO, and the control group was added with ddH in the same amount as the inhibitor solvent DMSO in the experimental group ₂ O treatment, then adding bacterial wilt bacteria to the experimental group and the control group and making the bacterial wilt bacteria OD in the culture solution ₆₀₀ The results of daily observation of arabidopsis thaliana after long-day cultivation at 28 ℃ and recording of the disease progression of bacterial wilt are shown in fig. 3, and the experimental group is ill, and the control group is not ill. In addition, the same effects as those of the 5. Mu.M kinase inhibitor described above can be achieved with the 10nM and 10. Mu.M kinase inhibitor.

Example 5

Phenotypic identification of kinase inhibitor Staurosporine treatment: 110g of soil is added into each small pot, 3 wild type Yunyan 87 seeds are respectively placed into each small pot, 5 repetitions are respectively carried out, 50mL of soil is covered, and after the seeds germinate, the seedlings with uneven growth vigor are pulled out, so that a tobacco seedling is left. After the plants grow up, a knife is cut into the root-approaching soil, 25mL of solution containing 5 mu M of kinase inhibitor Staurosporine is irrigated for 1 day, and 5mL of solution containing OD is added ₆₀₀ Pseudomonas solanacearum cqps=0.1; control group was irrigated with 25ml ddH ₂ O, after 1 day, 5mL of the mixture containing OD was added ₆₀₀ Ralstonia solanacearum cqps=0.1. And observing the growth condition of the tobacco after 10 days, and comparing the infection phenotype of the ralstonia solanacearum of the control group and the experimental group. Figure 4 illustrates that treatment with the kinase inhibitor Staurosporine also helps tobacco resist ralstonia solanacearum.

Claims

1. Use of a kinase inhibitor Staurosporine in improving resistance to tobacco, tomato and capsicum bacterial wilt.

2. The use according to claim 1, wherein the kinase inhibitor Staurosporine is Staurosporine as active ingredient.

3. The use according to claim 2, wherein the concentration of staurosporine is 10nM-10 μm.

4. Use according to claim 3, characterized in that the concentration of staurosporine is 5 μm.