CN118325926A - Application of chilo suppressalis salivary protein disulfide isomerase in improving tobacco resistance and method thereof - Google Patents

Abstract

The invention discloses an application of chilo suppressalis salivary protein disulfide isomerase in improving tobacco resistance and a method thereof, belonging to the technical field of agricultural science. The method specifically comprises the steps of finding a coding region sequence of the gene from a Chilo suppressalis genome according to a Chilo suppressalis salivary protein group database, constructing the coding region sequence into an over-expression vector suitable for plant expression, and obtaining a tobacco plant with transient over-expression of disulfide isomerase CsPDI by using an agrobacterium liquid infiltration leaf method. The insect resistance measurement such as cell death, ROS burst, jasmonic acid accumulation and the like is carried out on the strain. The results show that: disulfide isomerase CsPDI can obviously induce plant cell death, ROS burst, jasmonic acid accumulation and other resistance reactions after being expressed in tobacco, and positively regulate plant insect resistance. Therefore, the resistant variety can be cultivated by introducing the gene, and has great application value for plant breeding.

Description

Application and method of Chilo suppressalis salivary protein disulfide isomerase in improving tobacco resistance

Technical Field

The invention belongs to the field of agricultural science and technology, and relates to the application and method of disulfide bond isomerase CsPDI of salivary protein gene of Chilo suppressalis in inducing tobacco to produce resistance.

Background technique

The protein disulfide isomerase family (PDIs) belongs to the thioredoxin superfamily and is a multifunctional enzyme that is ubiquitous in eukaryotes. It is highly conserved in evolution and consists of an N-terminal signal peptide, four domains (a, b, b' and a') and a C-terminal endoplasmic reticulum retention signal peptide. The a and a' domains have a high homology with the thioredoxin structure (Thioredoxin, Trx), contain a conserved CGHC active site, and can catalyze the oxidation and reduction of disulfide bonds (Appenzeller-Herzog C and Ellgaard L. The human PDI family:Versatility packed into a single fold. BBA-Mol Cell Res. 2008, 1783: 535~548); the b and b' domains have no homology with the thioredoxin structure and can participate in the isomerization of disulfide bonds. PDIs play a key role in catalyzing the oxidative folding of nascent peptide chains in the endoplasmic reticulum, degradation and transport of endoplasmic reticulum-associated proteins and maintaining calcium homeostasis (Ding X, Lv ZM, Zhao Y, Min H, Yang WJ. MTH1745, a protein disulfideisomerase-like protein from thermophilic archaea, Methanothermobacterthermoautotrophicum involving in stress response. Cell Stress Chaperon. 2008,13: 239~246; and Ondzighi CA, Christopher DA, Cho EJ, Cho EJ, Chang SC, Staehelin

LA. Arabidopsis protein disulfide isomerase-5 inhibits cysteineproteases during trafficking to vacuoles before programmed cell death of theendothelium in developing seeds. Plant Cell. 2008, 20: 2205~2220).

With the understanding of plant-insect interactions and the development of biotechnology, the development of plant immune inducers and breeding of new insect-resistant varieties using insect saliva elicitors to induce broad-spectrum immunity is expected to become an important development direction for efficient green insect control. However, there are no reports on the research on the disulfide isomerase elicitor in the saliva of the Chilo suppressalis to induce plant resistance. This application discloses for the first time the expression of disulfide isomerase in tobacco and the induction of tobacco resistance, which has important application value for plant breeding.

Summary of the invention

In order to solve the above defects and shortcomings in the prior art, the present invention provides an application and method of Chilo suppressalis salivary protein disulfide isomerase in tobacco resistance. The Chilo suppressalis salivary protein disulfide isomerase CsPDI is expressed in tobacco leaves, which can effectively induce tobacco resistance.

To solve the above technical problems:

The present invention provides an application of a salivary protein disulfide isomerase CsPDI of a striped stem borer in improving tobacco resistance. The salivary protein disulfide isomerase CsPDI of a striped stem borer is expressed in tobacco leaves, and typical tests such as inducing plant cell death, ROS burst and jasmonic acid accumulation confirm that the salivary protein disulfide isomerase CsPDI of a striped stem borer can effectively induce tobacco resistance.

Furthermore, the nucleotide sequence of disulfide isomerase CsPDI is shown in SEQ ID NO.1.

Furthermore, the amino acid sequence of disulfide isomerase CsPDI is shown in SEQ ID NO.2.

The present invention also provides an overexpression vector pBINplus-GFP-CsPDI containing disulfide isomerase CsPDI, and a cell expressing the overexpression vector pBINplus-GFP-CsPDI. The details are as follows:

Firstly, the disulfide isomerase CsPDI and green fluorescent protein GFP were fused and constructed into the vector pBINplus to obtain the overexpression vector pBINplus-GFP-CsPDI. Then, the overexpression vector pBINplus-GFP-CsPDI was transformed into the competent cells of Agrobacterium, cultured and expanded, and the bacteria were obtained by centrifugation and resuspended in the infection solution. Finally, the infection solution was used to infiltrate tobacco leaves for transient overexpression of the disulfide isomerase CsPDI.

The present invention also provides a method for improving tobacco resistance, which is to express Chilo suppressalis salivary protein disulfide isomerase CsPDI in tobacco. The specific process is as follows:

S1 constructs an overexpression vector containing disulfide bond isomerase CsPDI: the disulfide bond isomerase CsPDI and green fluorescent protein GFP are fused to construct an overexpression vector PbinPLUS-GFP-CsPDI;

S2: electroporating the above overexpression vector into GV3101 Agrobacterium competent cells for cultivation;

S3: Infect tobacco leaves with the GV3101 Agrobacterium competent cells obtained in step S2 to obtain resistant tobacco plants.

The beneficial technical effects achieved by the present invention are as follows: the present invention discloses for the first time an application of a disulfide isomerase CsPDI of a striped stem borer in improving the resistance of tobacco plants. Specifically, the coding region sequence of the gene is found from the genome of the striped stem borer based on the salivary proteome database of the striped stem borer, and is constructed into an overexpression vector suitable for plant expression, and a tobacco plant transiently overexpressing the disulfide isomerase CsPDI is obtained by infiltrating leaves with Agrobacterium liquid. Insect resistance tests such as cell death, ROS burst, and jasmonic acid accumulation are performed on the plant. The results show that after the disulfide isomerase CsPDI is expressed in tobacco, it can significantly induce resistance reactions such as plant cell death, ROS burst, and jasmonic acid accumulation, and positively regulate the insect resistance of the plant. Therefore, resistant varieties can be cultivated by introducing the gene, which has great application value for plant breeding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a picture of the disulfide isomerase CsPDI gene expressed in tobacco, wherein 1A is a fluorescence photo, 1B is a bright field photo, and 1C is a combined fluorescence and bright field photo;

Figure 2 is the detection of resistance response indicators after transient overexpression of disulfide bond isomerase CsPDI in tobacco; among them, 2A is cell death, 2B is ROS burst, 2C is the accumulation of salicylic acid pathway key gene NbPR1 , 2D is the accumulation of salicylic acid pathway key gene NbPR2 , 2E is the accumulation of jasmonic acid pathway key gene NbPR3 , 2F is the accumulation of jasmonic acid pathway key gene NbPR4; ** indicates that there is a very significant difference between the treatment group and the control group;

FIG3 is a diagram showing the functional region causing plant cell death determined by a CsPDI -deficient mutant of disulfide isomerase; "+" indicates that it can cause cell death, and "-" indicates that it cannot cause cell death;

Figure 4 shows that transient overexpression of disulfide isomerase CsPDI in tobacco significantly reduced insect fitness performance, wherein 4A is a photo of the feeding tendency of fall armyworm and aphids, 4B is the number of fall armyworm on tobacco leaves overexpressing green fluorescent protein GFP and disulfide isomerase CsPDI , and 4C is the number of aphids on tobacco leaves overexpressing green fluorescent protein GFP and disulfide isomerase CsPDI ; ** indicates a very significant difference between the treatment group and the control group.

Detailed ways

The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

The present invention is further described below in conjunction with the accompanying drawings and embodiments.

Example 1 Expression of disulfide isomerase CsPDI in tobacco

(1) Construction of an overexpression vector containing the target gene: Design primers (upstream primer and downstream primer) of the target gene disulfide isomerase CsPDI (nucleotide sequence as shown in SEQ ID NO.1), and clone the PCR fragment. The PCR reaction system is 25ul of Tks Gflex™ DNA Polymerase buffer, 1ul of upstream and downstream primers, 1ul of C. suppressalis cDNA, 1ul of TksGflex™ DNA Polymerase, and 21ul of ultrapure water. The PCR reaction program is 94℃ for 3min, 98℃ for 10s, 60℃ for 30s, 72℃ for 60s, and 72℃ for 7min.

The upstream primer sequence is as shown in SEQ ID NO.3: AGCTGTACAAGGGTACCCCCGGGATGGACGACGTTGCCACAGA

The downstream primer sequence is as shown in SEQ ID NO.4: GACTCTAGTTCATCTAGAGGATCCTTATAACTCGTCTCTGGTTGGTGTT

According to the In-Fusion HD cloning kit (Takara, catalog number: 639633) instructions, the gel recovery product obtained by the PCR product was fused with the green fluorescent protein GFP gene through the double restriction sites BamH1 and Saml to construct the vector PbinPLUS-GFP to obtain the disulfide isomerase CsPDI overexpression vector PbinPLUS-GFP-CsPDI. After sequencing, the overexpression vector with correct sequencing was selected and transformed into the competent cells of GV3101 Agrobacterium by electroporation.

(2) Infection of Nicotiana benthamiana leaves: Pick the Agrobacterium liquid containing the correct overexpression vector for expansion culture, centrifuge the sediment, resuspend it in the infection solution (final concentration is 0.5M morpholineethanesulfonic acid, 1M magnesium chloride, 100mM acetosyringone), and adjust the concentration to _OD600 = 0.8. Let it stand in the dark for 2 hours, and infiltrate the back of the tobacco leaves with a disposable syringe to obtain resistant tobacco plants. After 24 hours, observe GFP fluorescence under a laser confocal microscope, and qualitatively confirm the expression of the disulfide bond isomerase CsPDI gene in tobacco by the intensity and range of fluorescence in the field of view, as shown in Figure 1. As can be seen from Figure 1, the disulfide bond isomerase CsPDI gene can be normally and efficiently expressed in tobacco, and the expression site is in the cytoplasm and nucleus.

Example 2 Resistance detection after transient expression of disulfide isomerase CsPDI in tobacco

(1) Observation of cell death symptoms: The tobacco leaves obtained in Example 1 were cultured for 3-5 days to observe the cell death phenomenon in the injection area, and the results are shown in Figure 2. Among them, 2A is cell death. It can be seen that after expressing the disulfide bond isomerase CsPDI gene, the infected part turned white, indicating that cell death occurred; while after expressing the green fluorescent protein GFP gene, the infected part did not change significantly. It can be seen that the disulfide bond isomerase CsPDI significantly induced the tobacco to produce a resistance reaction to cell death.

(2) Detection of ROS burst: The tobacco leaves obtained in Example 1 were used. After 16 hours of expression, the leaves of the treatment group were taken out and immersed in a PBS buffer solution (pH=7.0) with a final concentration of 1 mg/ml 3,3'-diaminobenzidine, and cultured in the dark overnight until brown spots appeared on the leaves. After that, the leaves were immersed in anhydrous ethanol and boiled to remove the green color of the leaves, and then observed and photographed. The results are shown in Figure 2. Among them, 2B is the ROS burst. It can be seen that after expressing the disulfide isomerase CsPDI gene, ROS burst obviously appeared in the infected area; while after expressing the green fluorescent protein GFP gene, there was no obvious change in the infected area. It can be seen that the disulfide isomerase CsPDI significantly induces tobacco to produce a resistance response to ROS burst.

(3) Detection of jasmonic acid and salicylic acid accumulation: The expression levels of jasmonic acid-related genes NbPR3 and NbPR4, as well as salicylic acid-related genes NbPR1 and NbPR2 were detected; leaves from the injection area were cut at 24 and 48 h, quickly frozen in liquid nitrogen, and RNA was extracted using the RN38-EASYspin Plus Plant RNA Rapid Extraction Kit (Beijing Aidelai Biotechnology Co., Ltd., Catalog No.: RN3802). The biological replicates were repeated three times, and each biological replicate consisted of a mixed sample of 5 leaves. After reverse transcription of the RNA samples, quantitative PCR was performed using the TB Green Premix Ex Taq ^TM II Kit produced by Takara (TaKaRa, RR820A). The quantitative PCR primer sequences are shown in Table 1: SEQ ID NO.5-SEQ ID NO.14. Leaves from the GFP injection area were used as controls, and the relative mRNA expression levels of the genes were calculated using the 2- ^ΔΔCt method. The results are shown in Figure 2. Among them, 2C is the accumulation of NbPR1 , a key gene in the salicylic acid pathway, 2D is the accumulation of NbPR2 , a key gene in the salicylic acid pathway, 2E is the accumulation of NbPR3 , a key gene in the jasmonic acid pathway, and 2F is the accumulation of NbPR4, a key gene in the jasmonic acid pathway. From 2C-2F, we can see that at 12h and 48h of CsPDI expression, the expression levels of NbPR3 and NbPR4 genes related to jasmonic acid signals were significantly higher than those in the GFP expression group, while the expression levels of NbPR1 and NbPR2 genes related to salicylic acid signals were significantly lower than those in the GFP expression group.

Table 1 Quantitative PCR primer sequences

Example 3 Determination of the functional region causing plant cell death

The four domains a, b, b' and a' of CsPDI were expressed by fusion with GFP protein alone or in combination by segmental deletion. After 24 hours, the expression of related genes in tobacco leaves was determined by laser confocal microscopy. The symptoms and intensity of cell death were then observed for 7 consecutive days. The results are shown in Figure 3: a-b-b'-a', b-b'-a', and b-b' can all cause cell death, while b'-a' and a' cannot cause cell death. This provides a research basis for inducing resistance by directly infiltrating plants with synthetic peptides.

Example 4 Effect of plants overexpressing disulfide isomerase CsPDI on insect feeding preference

The tobacco leaves obtained in Example 1 were used. After 20 hours of expression, CsPDI-GFP and GFP leaves were cut and placed in pairs in a culture dish. Eight 2nd-instar chewing insects, fall armyworms, or 15 piercing-sucking insects, tobacco aphids, which used tobacco as a host, were randomly selected and placed in the center of each pair of treated leaves. The selectivity of insects to leaves was recorded after 24 hours and 48 hours of feeding, and the results are shown in Figure 4. Among them, 4A is a photo of the feeding tendency of fall armyworms and aphids. From 4A, it can be seen that the number of fall armyworms and aphids on tobacco leaves overexpressing green fluorescent protein GFP is greater, and the leaves are seriously fed; while the number of fall armyworms and aphids on tobacco leaves overexpressing disulfide isomerase CsPDI is significantly less, and the leaves are only slightly fed. 4B and 4C respectively counted the number of fall armyworm and aphids on tobacco leaves overexpressing green fluorescent protein GFP and disulfide isomerase CsPDI , further confirming that fall armyworm and aphids prefer to feed on tobacco leaves expressing green fluorescent protein GFP, which shows that tobacco leaves expressing disulfide isomerase CsPDI show significant insect resistance.

The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims (8)

1. Use of chilo suppressalis salivary protein disulfide isomerase CsPDI in improving resistance of tobacco.

2. The use according to claim 1, characterized in that: the nucleotide sequence of disulfide isomerase CsPDI is shown in SEQ ID NO. 1.

3. The use according to claim 1, characterized in that: the amino acid sequence of disulfide isomerase CsPDI is shown in SEQ ID NO. 2.

4. An over-expression vector comprising the disulfide isomerase CsPDI according to any one of claims 2 or 3.

5. A cell expressing the over-expression vector of claim 4.

6. Use of the overexpression vector according to claim 4, or the cell according to claim 5, for inducing resistance in tobacco.

7. A method of increasing tobacco resistance, comprising: expressing the Chilo suppressalis salivary protein disulfide isomerase CsPDI in tobacco.

8. The method for improving tobacco resistance according to claim 7, wherein the specific process is as follows:

S1, constructing an overexpression vector containing disulfide isomerase CsPDI: the disulfide isomerase CsPDI and the green fluorescent protein GFP are fused and then are constructed into an overexpression vector PbinPLUS-GFP;

S2, performing electric shock transformation on the over-expression vector to GV3101 agrobacterium competent cells for culture;

S3, infecting tobacco leaves with the GV3101 agrobacterium competent cells obtained in the step S2 to obtain a resistant tobacco plant.