阅读说明：本技术 杀叶螨蛋白bvp8及其应用 (Tetranychus-killing protein BVP8 and application thereof ) 是由 刘晓艳 闵勇 陈凌 朱镭 邱一敏 饶犇 周荣华 龚艳 廖先清 陈伟 邱春富 石 于 2021-07-05 设计创作，主要内容包括：本发明属于农业微生物学技术领域,具体涉及杀叶螨蛋白BVP8及其应用。所述蛋白为SEQ ID NO.2所示。BVP8蛋白对二斑叶螨半致死浓度为12.98μg/mL,对柑橘全爪螨的半致死浓度为33.45μg/mL,对朱砂叶螨的半致死浓度为26.32μg/mL,对二斑叶螨的卵具有抑制孵化及裂解的作用,72 h的卵裂解率为75.86%。该蛋白为新型的杀螨剂制备提供了新的选择性。(The invention belongs to the technical field of agricultural microbiology, and particularly relates to a tetranychidae killing protein BVP8 and application thereof. The protein is shown in SEQ ID NO. 2. The semi-lethal concentration of the BVP8 protein to Tetranychus urticae is 12.98 mug/mL, the semi-lethal concentration to Panonychus citri is 33.45 mug/mL, the semi-lethal concentration to Tetranychus cinnabarinus is 26.32 mug/mL, the incubation and cracking inhibiting effects are achieved on eggs of the Tetranychus urticae, and the egg cracking rate is 75.86% after 72 hours. The protein provides new selectivity for the preparation of novel acaricides.)

1. A tetranychid mite killing protein is shown in SEQ ID NO. 2.

2. A gene for coding the protein shown in SEQ ID NO. 2.

3. The gene of claim 2, which is represented by SEQ ID No. 1.

4. Use of a tetranychicide protein according to claim 1 for preparing a tetranychicide.

5. Use of the tetranychicidal protein of claim 1 in the preparation of a hatching inhibitor for spider mite eggs.

6. The use according to claim 4 or 5, wherein the tetranychus urticae is tetranychus urticae, panonychus citri or tetranychus cinnabarinus.

Technical Field

The invention belongs to the technical field of agricultural microbiology, and particularly relates to a tetranychidae killing protein BVP8 and application thereof.

Background

Spider mites are important pest mites in agricultural production. Is harmful to cotton, grains, fruit trees, forest trees and ornamental plants. Tetranychus urticae (tetranychus urticae) is one of ten pests in China. The tetranychus urticae can pierce 18-22 plant cells per minute. The panonychus citri is an important pest mite of citrus trees, the panonychus ulmi and tetranychus crataegi are important pest mites of fruit trees in northern China, and the fruit yield can be reduced by 1/3-2/3 in case of serious damage. The wheat rock mites spread throughout the wheat producing area of the whole country, and when the wheat rock mites are seriously harmful, the wheat grains can not be harvested. In the cotton field, various tetranychids of cotton are harmful, the yield can be reduced by more than 30 percent, and even cotton plants are completely scorched.

Temperature is an important factor affecting the growth and decline of the population of spider mites. The temperature range and suitable temperature for spider mite activity will often vary from species to species. The high-temperature active species, such as the jatropha pomonensis, is suitable for the growth temperature of 25-28 ℃; the low-temperature activity type, such as the varroa destructor, is 21-24 ℃. Most spider mites prefer dry climate, and the relative humidity is preferably 40-70%. Thus, dry and hot weather often leads to rampant spider mites. The breeding mode of spider mites is mainly amphoteric reproduction and parthenogenesis. The species such as the lichen mites only refer to female mites, and the carpesium mites are parthenogenesis. Species such as Tetranychus have male and female sex, and can perform sexual reproduction and parthenogenesis, that is, unfertilized female mites only produce male offspring.

The method for preventing and controlling tetranychus urticae is mainly divided into agricultural prevention and control, biological prevention and control and chemical prevention and control. The agricultural control comprises cleaning the garden in winter, cleaning the diseased leaves and burning the diseased leaves intensively, and reducing the overwintering number of the pest mites; strengthen the management of fertilizer and water, improve the microclimate of the orchard, and the like. The biological control comprises the control by utilizing green and safe means such as natural enemies, biological acaricides and the like. Chemical control is mainly carried out by using chemical acaricide and other medicaments. In recent years, because the variety of acaricides is lack of substitution, the acaricides are repeatedly used in large quantity, the generation of resistance to harmful mites is accelerated, the mite damage is more and more serious, and great economic loss is caused to growers.

At present, most of acaricides are chemical substances or macromolecular antibiotics which are insoluble in water internationally, reports on small molecular protein substances are few, and based on the background, protein preparations for preventing and treating spider mites need to be developed, so that the acaricides have important production practice significance in the field of biological control of spider mites.

Disclosure of Invention

The invention aims to provide a tetranychid mite killing protein which is shown in SEQ ID No. 2.

The invention also aims to provide the application of the tetranychid-killing protein in preparing a tetranychid-killing agent.

In order to achieve the purpose, the invention adopts the following technical measures:

a tetranychid mite killing protein which is shown in SEQ ID NO. 2; the gene for coding the protein shown in SEQ ID NO.2 also belongs to the protection scope of the invention, and the preferred gene is shown in SEQ ID NO. 1.

The above proteins, which are expressed in a conventional manner in the art, such as by prokaryotic, eukaryotic expression, or by direct synthesis, are all within the scope of the present invention

The application of the tetranychus-killing protein in preparing the tetranychus-killing agent comprises the step of preparing the tetranychus-killing agent by taking the protein shown in SEQ ID NO.2 as one of effective components or the only effective component; the tetranychicide protein can also be used as a hatching inhibitor for eggs of tetranychidae.

In the above application, preferably, the tetranychus urticae is tetranychus urticae koch, panonychus citri or tetranychus cinnabarinus.

Compared with the prior art, the invention has the following characteristics:

the invention reports a new protein for killing tetranychus urticae and the acaricidal activity thereof for the first time, and provides new selectivity for preparing a novel acaricide.

The acaricide containing BVP8 protein has the advantages of high efficiency, low toxicity and environmental friendliness.

Drawings

FIG. 1 is an SDS-PAGE electrophoretic analysis of purified BVP8 protein.

FIG. 2 is a graph showing the effect of BVP8 protein on Tetranychus urticae egg.

Detailed Description

The experimental procedures in the following examples are reported as conventional procedures in microbiology unless otherwise specified. Such reagents or materials, unless otherwise specified, are conventional in the art.

The BVP8 protein related to the present invention can be obtained by conventional methods in the art, such as prokaryotic expression, commercial synthesis, etc. The invention takes prokaryotic expression BVP8 protein as an example to explain the mite inhibiting function, and the protein obtained by other methods can also perform the same function.

Example 1:

obtaining of acaricidal BVP8 protein:

1) BVP8 protein gene fragment (Biotechnology engineering (Shanghai) Co., Ltd.) was synthesized artificially according to the sequence shown in SEQ ID NO.1 (encoding the protein shown in SEQ ID NO. 2) and ligated to the E.coli expression vector pet28a plasmid to construct recombinant expression plasmid pet28a-BVP 8.

2) Expression and purification of acaricidal BVP8 protein gene in escherichia coli BL21

In order to express acaricidal BVP8 protein in a large amount, the recombinant expression plasmid pet28a-BVP8 carrying the coding sequence is transformed into Escherichia coli BL21 to obtain recombinant bacteria BL21/pet28a-BVP 8. The recombinant strain was inoculated into 5mL of LB liquid medium, shake-cultured at 37 ℃ until OD600 became 0.6, and then 1.0mmol/L isopropyl-B-D thiogalactoside (i.e., IPTG, available from Sigma) was added and induced at 30 ℃ for 3 hours. 50mL of the induced culture of the recombinant bacterium BL21/pet28a-BVP8 for 3 hours was centrifuged at 12000rpm for 30 seconds to collect the cells, the cells were disrupted by ultrasonication (technical parameters: 300W, disruption for 30 seconds, and pause for 30 seconds), and then centrifuged at 12000rpm for 15 minutes to collect the supernatant, which was then filtered through a 0.4 μm pore filter to remove impurities, and each protein was purified by affinity chromatography using GST fusion protein. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection is carried out on the final purified product, the result is shown in figure 1, the molecular weight of the purified protein is compared with the molecular weight standard of the protein, the estimated molecular weight is 9kDa, and the molecular weight is basically consistent with the estimated molecular weight of the protein BVP8, the size of which is 8.34kDa, so that the BVP8 protein is successfully expressed in escherichia coli BL 21.

Example 2:

1) BVP8 protein is used for killing Tetranychus urticae (Tetranychus urticae):

reference is made to the standard method recommended by FAO (food and agriculture organization of the united nations) for the determination of mites-the glass slide immersion method. The double-sided adhesive tape is cut into 2-3cm long, the double-sided adhesive tape is attached to one end of a microscope slide, paper pieces on the adhesive tape are removed by tweezers, female adult mites which are consistent in size, bright in body color and lively in action are selected by a zero-number brush pen, the backs of the female adult mites are adhered to the double-sided adhesive tape (paying attention to the fact that the female adult mites do not stick to the mite feet, the mite beards and a mouth tool), 4 lines of the double-sided adhesive tape are adhered to each piece, and 10 heads of the double-sided adhesive tape are adhered to each line. Placing in a biochemical incubator with the temperature of 25 ℃ and the relative humidity of about 85% for 4h, observing by using binoculars, and removing dead or inactive individuals. Diluting the medicament with water for 5-7 concentrations on the basis of pre-test, soaking one end of the glass slide with the mites into the medicinal liquid, slightly shaking for 5s, taking out, and quickly sucking away the mites and the redundant medicinal liquid around the mites with absorbent paper. Placing in the biochemical incubator, and inspecting the result by using binoculars after 24 h. The mites were touched with a brush pen and died if they were immobile. Each concentration was repeated 3 times, and the control was made by soaking in clean water. The results of the test on Tetranychus urticae by BVP8 protein suspension according to the above experimental procedures are shown in Table 1 below, and are 12.98. mu.g/mL. LC (liquid Crystal)₅₀The calculation of the values was obtained by calculation using the span 19.0 data processing software.

TABLE 1 acaricidal Activity of BVP8 protein on Tetranychus urticae

2) BVP10 protein killed Panonychus citri (panocychs citri):

reference is made to the standard method recommended by FAO (food and agriculture organization of the united nations) for the determination of mites-the glass slide immersion method. The double-sided adhesive tape is cut into 2-3cm long, the double-sided adhesive tape is attached to one end of a microscope slide, paper pieces on the adhesive tape are removed by tweezers, female adult mites which are consistent in size, bright in body color and lively in action are selected by a zero-number brush pen, the backs of the female adult mites are adhered to the double-sided adhesive tape (paying attention to the fact that the female adult mites do not stick to the mite feet, the mite beards and a mouth tool), 4 lines of the double-sided adhesive tape are adhered to each piece, and 10 heads of the double-sided adhesive tape are adhered to each line. Placing in a biochemical incubator with the temperature of 25 ℃ and the relative humidity of about 85% for 4h, observing by using binoculars, and removing dead or inactive individuals. Diluting the medicament with water for 5-7 concentrations on the basis of pre-test, soaking one end of the glass slide with the mites into the medicinal liquid, slightly shaking for 5s, taking out, and quickly sucking away the mites and the redundant medicinal liquid around the mites with absorbent paper. Placing in the biochemical incubator, and inspecting the result by using binoculars after 24 h. The mites were touched with a brush pen and died if they were immobile. Each concentration was repeated 3 times, and the control was made by soaking in clean water. The results of the test on Panonychus citri using BVP8 protein suspension according to the above experimental procedures are shown in Table 2 below as 33.45. mu.g/mL. LC (liquid Crystal)₅₀The calculation of the values was obtained by calculation using the span 19.0 data processing software.

TABLE 2 acaricidal Activity of BVP8 protein on Panonychus citri

3) According to the experimental procedures, the result of the bioassay of the BVP8 protein suspension on Tetranychus cinnabarinus is shown in Table 3 below, and is 26.32. mu.g/mL. LC (liquid Crystal)₅₀The calculation of the values was obtained by calculation using the span 19.0 data processing software.

TABLE 3 miticidal Activity of BVP8 protein on Tetranychus cinnabarinus

4) Inhibition effect of BVP8 protein on tetranychus urticae egg

Taking out the leaf of Tetranychus urticae in the egg-laying period, and flushing away adult mites and nymphs under running water. The absorbent paper absorbs the water of the leaves. And (4) lightly sticking the eggs by using a glass slide stuck with double-sided adhesive, observing and counting under a microscope, and immersing the counted eggs into a sample to be detected for three seconds and taking out. Then placing the eggs into a constant temperature incubator with the temperature of 25 ℃, observing the effect of the BVP8 protein on the eggs of the tetranychus urticae under a microscope after 24 hours, 48 hours and 72 hours respectively, counting the eggs with different forms and taking pictures.

The results show that the BVP8 protein has an inhibiting effect on the eggs of Tetranychus urticae, can inhibit the hatching of the eggs, the eggs begin to shrink and gradually crack after 24h, and the cracking rate of the eggs at 48h and 72h is 22.41 percent and 75.86 percent respectively (Table 4, figure 2).

TABLE 4 inhibitory Effect of BVP8 protein on Tetranychus urticae egg

Remarking: completely filling eggs, yellowish in color, and waiting for hatching; + + normal eggs, partial hatching; b, hatching eggs; + + + + +, eggs shriveling, not hatching; + + + ++, the egg is lysed.

Sequence listing

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