BVP8 PROTEIN FOR KILLING TETRANYCHID MITES AND USE THEREOF

Abstract

A BVP8 protein for killing tetranychid mites and use thereof are provided. The protein is as set forth in SEQ ID NO. 2. The BVP8 protein has a median lethal concentration of 12.98 μg/mL against Tetranychus urticae, 33.45 μg/mL against Panonychus citri, and 26.32 μg/mL against Tetranychus cinnabarinus, and shows an inhibitory effect against the hatching and cleavage of Tetranychus urticae eggs, with the egg cleavage rate of 75.86% after 72 h. The protein provides a new option for the preparation of a novel miticide.

Claims

1. A method for controlling tetranychid mites, comprising spraying a biocide comprising a protein of SEQ ID NO:2 on tetranychid mites.

2. The method according to claim 1, wherein the tetranychid mites are Tetranychus urticae, Panonychus citri, or Tetranychus cinnabarinus.

3. The method according to claim 1, wherein the protein of SEQ ID NO:2 has a median lethal concentration of 12.98 μg/mL against Tetranychus urticae.

4. The method according to claim 1, wherein the protein of SEQ ID NO:2 has a median lethal concentration of 33.45 μg/mL against Panonychus citri.

5. The method according to claim 1, wherein the protein of SEQ ID NO:2 has a median lethal concentration of 26.32 μg/mL against Tetranychus cinnabarinus.

6. A method for inhibiting hatching of tetranychid mite eggs, comprising spraying a biocide containing a protein as set forth in SEQ ID NO:2 to eggs of tetranychid mites.

7. The method according to claim 6, wherein the tetranychid mites are Tetranychus urticae, Panonychus citri, or Tetranychus cinnabarinus.

8. The method according to claim 6, wherein a cleavage rate is 75.86% or less at 72 hours after spraying the biocide for Tetranychus urticae eggs.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0016] FIG. 1 shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified BVP8 protein of the present invention.

[0017] FIGS. 2A to 2F show the effects of the BVP8 protein of the present invention against Tetranychus urticae mite eggs, where FIG. 2A to 2C show the effects of control group after 24 hours, 48 hours, and 72 hours, respectively, after administration; and FIGS. 2D to 2F show the effects of the BVP8 protein of the present invention after 24 hours, 48 hours, and 72 hours, respectively after administration.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Experimental methods in the embodiments below are all conventional microbiological operation methods that have been reported, unless otherwise specified. Reagents or materials as mentioned are those used in conventional solutions in the art, unless otherwise specified.

[0019] The BVP8 protein mentioned in the invention can be prepared by a conventional method, such as prokaryotic expression and commercial synthesis, in the art. The present invention illustrates the mite inhibition function of a prokaryotically expressed BVP protein, by way of example. This protein derived by other means may also exert the same effect.

Embodiment 1

Preparation of Miticidal Protein BVP8

[0020] (1) Based on a sequence (encoding a protein as set forth in SEQ ID NO. 2) as set forth in SEQ ID NO.1, a BVP8 protein gene fragment (from Sangon Biotech (Shanghai) Co., Ltd.) was artificially synthesized, and then attached to a pET 28a plasmid of Escherichia coli expression vector to construct a recombinant expression plasmid pET28a-BVP8.

(2) Expression and Purification of Miticidal BVP 8 Protein Gene in Escherichia coli BL21

[0021] To express the miticidal BVP8 protein at a high level, the above-mentioned recombinant expression plasmid pET28a-BVP8 carrying a coded sequence was transformed into Escherichia coli BL21 to prepare recombinant bacteria BL21/pET28a-BVP8. The recombinant bacteria were inoculated into 5 mL of Luria-Bertani (LB) liquid medium, and cultured in a shaker at 37° C. until OD600 was 0.6. Then, 1.0 mmol/L isopropyl-B-D-thiogalactoside (IPTG, from Sigma) was added for induced culturing for 3 h at 30° C. 50 mL of a 3-hour induced culture of the above-mentioned recombinant bacteria BL21/pET28a-BVP8 was centrifuged at 12,000 rpm for 30 s to collect bacterial cells. Bacteria cells were disrupted by ultrasonic waves (technical parameters: 300 W; 30 s; and 30 s interval), and then centrifuged at 12,000 for 15 min to obtain a supernatant, which was filtered with a filter membrane having a pore size of 0.45 μm to remove impurities. Proteins were purified by the affinity chromatography for His fusion proteins. A final purified product was detected by SDS-PAGE, with the results shown in FIG. 1. The purified proteins were aligned with a protein molecular weight marker to derive an estimated molecular weight of 9 kDa, which was basically in line with the molecular weight of 8.34 kDa as predicted for the BVP8 protein. This demonstrated the successful expression of the BVP8 protein of the invention in Escherichia coli B21.

Embodiment 2

(1) BVP8 Protein for Killing Tetranychus urticae

[0022] Referring to the standard method for determining pest mites, namely, the slide dipping method, recommended by Food and Agriculture Organization of the United Nations (FAO), a double-faced adhesive tape was cut into pieces of 2-3 cm long and stuck to one end of a microscope slide, and paper on the adhesive tape was removed with tweezers. Female adult mites with similar size, bright body color and high vitality were picked with a Chinese writing brush #0, and were stuck to the double-faced adhesive tape at backs (note: the feet, whiskers and mouthparts of the mites should not be stuck), with 4 lines stuck to each piece and 10 mites in each line. The mites were placed and left in a biochemical incubator with the temperature of 25 and the relative humidity of approx. 85% for 4 h, and then were observed with binoculars. Dead or inactive individuals were eliminated. An agent was diluted by 5-7 times with water based on a preliminary test. The end of the slide with the mites was dipped into the agent solution, shook gently for 5 s and then taken out. The mites as well as the excessive agent solution therearound were rapidly dried with absorbent paper. The slide was placed and left in the above-mentioned biochemical incubator. After 24 h, the results were checked with the binoculars. The bodies of the mites were lightly touched with the Chinese brush pen, and those without any motion of feet were considered to be dead. The test was repeated three times at each concentration, and the mites dipped in fresh water were additionally taken as a control. Following the experimental steps above, the bioassay result of a BVP8 protein suspension against Tetranychus urticae was shown in Table 1 below, which was 12.98 μg/mL. An LC.sub.50 value was calculated by using SPASS 19.0 data processing software.

TABLE-US-00001 TABLE 1 Miticidal activity of BVP8 protein against Tetranychus urticae Medial lethal Dose Mortality Logarithmic Probability Regression concentration (μg/mL) (%) dose unit (P + 5) equation (LC.sub.50, μg/mL) 165.5 56.7 2.219 5.170 Y = 4.7430 + 12.98 82.75 58.3 1.918 5.210 0.2308X (r = 33.1 62.5 1.520 5.320 0.5130) 16.55 44.4 1.219 4.859

(2) BVP8 Protein for Killing Panonychus citri

[0023] Referring to the standard method for determining pest mites, namely, the slide dipping method, recommended by Food and Agriculture Organization of the United Nations (FAO), a double-faced adhesive tape was cut into pieces of 2-3 cm long and stuck to one end of a microscope slide, and paper on the adhesive tape was removed with tweezers. Female adult mites with similar size, bright body color and high vitality were picked with a Chinese writing brush #0, and were stuck to the double-faced adhesive tape at backs (note: the feet, whiskers and mouthparts of the mites should not be stuck), with 4 lines stuck to each piece and 10 mites in each line. The mites were placed and left in a biochemical incubator with the temperature of 25 and the relative humidity of approx. 85% for 4 h, and then were observed with binoculars. Dead or inactive individuals were eliminated. An agent was diluted by 5-7 times with water based on a preliminary test. The end of the slide with the mites was dipped into the agent solution, shook gently for 5 s and then taken out. The mites as well as the excessive agent solution therearound were rapidly dried with absorbent paper. The slide was placed and left in the above-mentioned biochemical incubator. After 24 h, the results were checked with the binoculars. The bodies of the mites were lightly touched with the Chinese brush pen, and those without any motion of feet were considered to be dead. The test was repeated three times at each concentration, and the mites dipped in fresh water were additionally taken as a control. Following the experimental steps above, the bioassay result of a BVP8 protein suspension against Panonychus citri was shown in Table 2 below, which was 33.45 μg/mL. An LC.sub.50 value was calculated by using SPASS 19.0 data processing software.

TABLE-US-00002 TABLE 2 Miticidal activity of BVP8 protein against Panonychus citri Medial lethal Dose Mortality Logarithmic Probability Regression concentration (μg/mL) (%) dose unit (P + 5) equation (LC.sub.50, μg/mL) 165.5 76.9 2.591 2.219 Y = 3.5091 + 33.45 82.75 63.6 1.892 1.918 0.9780X (r = 33.1 45.5 1.591 1.520 0.9781) 16.55 41.7 1.289 1.219

[0024] (3) Following the experimental steps above, the bioassay result of a BVP8 protein suspension against Tetranychus cinnabarinus was shown in Table 3 below, which was 26.32 μg/mL. An LC.sub.50 value was calculated by using SPASS 19.0 data processing software.

TABLE-US-00003 TABLE 3 Miticidal activity of BVP8 protein against Tetranychus cinnabarinus Medial lethal Dose Mortality Logarithmic Probability Regression concentration (μg/mL) (%) dose unit (P + 5) equation (LC.sub.50, μg/mL) 165.5 97.2 2.219 6.918 Y = 1.8201 + 26.32 82.75 82.1 1.918 5.922 2.2390X (r = 33.1 59.5 1.520 5.239 0.9904) 16.55 34.3 1.219 4.596

(4) Inhibitory Effect of BVP8 Protein Against Tetranychus urticae Eggs

[0025] Leaf blades with Tetranychus urticae eggs at a full egg period were collected and flushed under running water to remove adult and nymph mites. Absorbent paper was used to absorb water on the leaf blades. The eggs were gently taken down by using a slide with double-faced tape stuck thereto, and observed and counted under a microscope. After counting, the eggs were dipped into a sample to be detected for 3 seconds and then taken out. Then, the eggs were placed into a constant-temperature incubator at 25° C., and observed under the microscope after 24 hr, 48 hr and 72 hr respectively in terms of the inhibitory effect of the BVP8 protein against the Tetranychus urticae eggs. The eggs having different forms were counted and photographed.

[0026] Results show that the BVP8 protein has an inhibitory effect on the Tetranychus urticae eggs, is capable of inhibiting the hatching of eggs, which start atrophy and are gradually cleaved after 24 hr, with the cleavage rates at 48 hr and 72 hr being 22.41% and 75.86% respectively (Table 4, FIGS. 2A to 2F).

TABLE-US-00004 TABLE 4 Inhibitory effect of BVP8 protein against Tetranychus urticae eggs 0 hr 24 hr 48 hr 72 hr Hatching Hatching Hatching Hatching Designation State rate (%) State rate (%) State rate (%) State rate (%) BVP8 + 0 ++++ 0 ++++ 0 +++++ 0 protein Fresh water + 0 ++ 17.86 +++ 50 +++ 83.93 as control Note: +: pale-yellow full eggs to be hatched; ++: normal eggs that have been hatched partially; +++: hatched eggs; ++++: unhatched shrunk eggs; +++++: cleaved eggs.