Application of rapamycin in improving the mosquito-killing activity of Bacillus thuringiensis mosquito-killing toxin

Abstract

The present invention relates to a new use of rapamycin, and in particular to the use of rapamycin in improving the mosquito-killing activity of Bacillus thuringiensis mosquito-killing toxin. The present invention is the first to apply rapamycin, an immunosuppressant in clinical medicine, to the study of microbial control of pathogenic mosquitoes, and finds that the addition of a low dose of rapamycin can significantly improve the mosquito-killing activity of Bacillus thuringiensis Cry4Ba toxin. The present invention provides an important basis for the subsequent development of new biological pesticides and related small molecule synergists, and is of great significance in improving the activity of microbial pesticides and reducing the cost of use. It also provides a new research direction for studying the insecticidal mechanism of biological control microorganisms represented by Bacillus thuringiensis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of rapamycin in improving the mosquito-killing activity of Bacillus thuringiensis mosquito-killing toxin. Background Art

[0002] Pathogenic mosquitoes (Aedes, Culex and Anopheles) are important pests that endanger human life and health. They spread a variety of virulent viruses and parasites such as malaria, Zika, West Nile fever and dengue fever through biting and blood sucking, causing hundreds of millions of cases and millions of deaths worldwide every year. Therefore, effective prevention and control of disease-carrying mosquitoes is a problem that needs to be solved urgently around the world. At present, the use of chemical insecticides is the main measure for the prevention and control of adult mosquitoes, but the large-scale use in recent years has led to a high level of drug resistance in mosquitoes. At the same time, chemical pesticides have certain toxicity to non-target organisms and cause ecological pollution. The development of efficient biological control strategies will be an important research and application direction at present and in the future. Among them, microbial mosquito-killing preparations prepared with Bacillus thuringiensis subspecies Israel as raw materials have been widely used worldwide due to their high safety, specificity and low resistance to resistance, and have been recommended by the World Health Organization. However, microbial control still has problems such as high cost, slow effect and weak stress resistance, which need to be further solved. Therefore, actively screening and developing an efficient enhancer plays an important role in promoting the development of Bacillus thuringiensis related products.

[0003] Rapamycin is a secondary metabolite secreted by soil Streptomyces. It was first used as an antifungal drug in medicine. With the deepening of research in recent years, it has been found that it has a highly effective immunosuppressive effect, and thus has been developed as a new type of immunosuppressant. Pharmacology shows that rapamycin can bind to the intracellular immunophilin FKBP12, thereby regulating the mTOR signaling pathway, further inhibiting cell growth and other physiological and biochemical effects. Studies have also revealed that rapamycin has development potential in many aspects such as anti-aging, anti-cancer and neuroprotection. In agricultural control, rapamycin can be mixed with fungal chemical fungicides to inhibit plant early blight. However, there are no reports on rapamycin in promoting the virulence of insecticidal microorganisms. The present invention uses rapamycin as an additive for a natural product microbial pesticide for the first time, and significantly improves the activity of Bacillus thuringiensis mosquito-killing toxin through low doses of rapamycin. Summary of the invention

[0004] The purpose of the present invention is to provide the use of rapamycin in improving the mosquito-killing activity of Bacillus thuringiensis mosquito-killing toxin.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] Application of rapamycin in improving the mosquito-killing activity of Bacillus thuringiensis mosquito-killing toxin.

[0007] Wherein, the structural formula of rapamycin is as follows:

[0008]

[0009] The specific application method of the rapamycin is: mixing the mosquito-killing toxin of Bacillus thuringiensis with rapamycin.

[0010] Wherein, the Bacillus thuringiensis mosquito-killing toxin is the Bacillus thuringiensis mosquito-killing toxin Cry4Ba protein.

[0011] Among them, the preferred specific application method is: adding Cry4Ba protein with a final concentration of 2 μg / ml to a 50 μM rapamycin solution and mixing evenly.

[0012] The significant advantages of the present invention are:

[0013] The present invention firstly finds that low-dose rapamycin can significantly improve the mosquito-killing activity of Bacillus thuringiensis Cry4Ba toxin. Under the combined action of low-dose rapamycin and semi-lethal dose of Cry4Ba toxin, the mortality rate of Aedes aegypti larvae in 4 hours is as high as 84±7.1%, and the mortality rate in 6 hours is as high as 96.9±3.7%; under the single action of semi-lethal dose of Cry4Ba toxin, the mortality rate of Aedes aegypti larvae in 4 hours is only 4.4±2.9%, the mortality rate in 6 hours is only 8.9±4.5%, the mortality rate in 12 hours is only 40.9±12.5%, and the mortality rate in 24 hours is only 57.8±10.2%; under the single action of rapamycin, all Aedes aegypti larvae survive within 24 hours. Therefore, low-dose rapamycin has a significant promoting effect on the mosquito-killing toxicity of Cry4Ba protein, which can not only significantly improve the mortality rate of larvae, but also significantly shorten the death time of larvae. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the synergistic effect of rapamycin on the killing of Aedes aegypti larvae by Bacillus thuringiensis Cry4Ba protein. DETAILED DESCRIPTION

[0015] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0016] The experimental materials involved in the present invention are as follows:

[0017] Rapamycin: Its structural formula is Purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0018] Preparation of mosquito-killing toxin Cry4Ba protein of Bacillus thuringiensis: 50 μL of recombinant Bacillus thuringiensis Bt-Cry4Ba was added to 5 ml LB liquid medium containing erythromycin at a final concentration of 10 μg / ml, and cultured overnight at 30°C. Then, the Bt-Cry4B culture solution was added to 1 / 2 LB liquid medium (containing 10 μg / ml erythromycin) at a ratio of 1 / 100, and cultured at 30°C, 200 r / min, and shaken for 4 days to release most of the crystal protein from the spores. The bacterial solution was collected by centrifugation at 4°C, 9500×g for 10 min, and the precipitate was washed twice with pre-cooled 1M NaCl solution and ultrapure water, respectively, and centrifuged at 4°C, 9500×g for 10 min. An appropriate amount of dissolution buffer (50mM Na2CO3, 1mM DTT, pH 10.5) was added and pipetted to mix well, and the solution pH was adjusted to 10.5 with 10M NaOH solution, and the solution was placed on ice and shaken slowly for 2-4 hours. Centrifuge at 4℃, 9500×g for 10min, aspirate the supernatant into a new centrifuge tube, adjust the pH to 4.6-4.8 with acetic acid, and white floccules will appear. Let stand at 4℃ for 1-4h until the crystal protein is completely precipitated and precipitated. Centrifuge at 4℃, 9500×g for 10min, remove the supernatant, and wash the precipitate twice with pre-cooled ultrapure water. Add appropriate amount of 50mM Na2CO3 to dissolve, and adjust the pH to 10.5, and gently shake on ice until the crystal protein is completely dissolved. Aliquot the protein and store at -80℃. Among them, the recombinant Bacillus thuringiensis Bt-Cry4Ba has been published in [Wang J, Yang X, He H, Chen J, Liu Y, Huang W, Ou L, Yang Z, Guan X, Zhang L, Wu S. Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins. Insects 2021; 12: 223. [PMID: 33807543 DOI: 10.3390 / insects12030223]].

[0019] Aedes aegypti larvae: provided by the Biopesticide Research Center of Fujian Agriculture and Forestry University, rearing conditions: temperature 26±1℃, humidity 83±3%, photoperiod 14h / 10h (day / night). Aedes aegypti larvae were fed with goldfish feed, and cotton blocks containing 10% sucrose solution were placed during the adult stage. Pregnant female mosquitoes sucked sterile defiberized newborn calf blood through a membrane feeding system to promote egg laying.

[0020] Bioassay: Wild strains of Aedes aegypti were reared to the early IV instar, 25 larvae were taken and transferred to 20 ml filtered water, and Cry4Ba crystal protein was added at final concentrations of 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 5 μg / ml, 10 μg / ml, and 18.75 μg / ml, and three biological replicates were performed. After 24 hours, the mortality of the larvae was counted, and the median lethal concentration (LC50) for the larvae was calculated using PoloPlus software to be 2.476±3.023 μg / ml.

[0021] Example 1

[0022] S1: dissolve rapamycin in DMSO to prepare a stock solution with a concentration of 10 mM; take 100 μl of the rapamycin stock solution and add it to 20 ml of pure water to obtain a rapamycin solution with a concentration of 50 μM.

[0023] S2: Add 25 4th instar larvae of Aedes aegypti to the 50 μM rapamycin solution in S1, and add the mosquito-killing toxin Cry4Ba protein of Bacillus thuringiensis at a final concentration of 2 μg / ml, and mix thoroughly.

[0024] S3: The mortality of larvae was observed at 1h, 2h, 3h, 4h, 6h, 12h, 18h and 24h after treatment, and each group of experiments was repeated 3 times.

[0025] Comparative Example 1

[0026] S1: Add 25 4th instar larvae of Aedes aegypti into 2 μg / ml of Bacillus thuringiensis mosquito-killing toxin Cry4Ba protein and mix thoroughly.

[0027] S2: The mortality of larvae was observed at 1h, 2h, 3h, 4h, 6h, 12h, 18h and 24h after treatment, and each group of experiments was repeated 3 times.

[0028] Comparative Example 2

[0029] S1: dissolve rapamycin in DMSO to prepare a stock solution with a concentration of 10 mM; take 100 μl of the rapamycin stock solution and add it to 20 ml of pure water to obtain a rapamycin solution with a concentration of 50 μM.

[0030] S2: Add 25 4th instar larvae of Aedes aegypti to the 50 μM rapamycin solution in S1 and mix thoroughly.

[0031] S3: The mortality of larvae was observed at 1h, 2h, 3h, 4h, 6h, 12h, 18h and 24h after treatment, and each group of experiments was repeated 3 times.

[0032] The results are as follows Figure 1As shown, in Example 1, under the action of low-dose rapamycin and semi-lethal dose of Cry4Ba protein, the mortality rate of Aedes aegypti larvae in 4h is as high as 84±7.1%, the mortality rate in 6h is as high as 96.9±3.7%, and almost all the test insects die; in Comparative Example 1, under the action of Bacillus thuringiensis mosquito-killing toxin Cry4Ba protein alone, the mortality rate of Aedes aegypti larvae in 4h is only 4.4±2.9%, the mortality rate in 6h is only 8.9±4.5%, the mortality rate in 12h is only 40.9±12.5%, and the mortality rate in 24h is only 57.8±10.2%; in Comparative Example 2, under the action of rapamycin alone, all the test insects survived within 24h. It can be seen that low-dose rapamycin has a significant promoting effect on the mosquito-killing toxicity of Cry4Ba protein, which can not only significantly increase the mortality rate of larvae, but also significantly shorten the death time of larvae.

[0033] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. The use of rapamycin in improving the mosquito killing activity of Bacillus thuringiensis mosquito killing toxin, characterized in that: The specific application method is as follows: add Bacillus thuringiensis mosquito-killing toxin with a final concentration of 2 μg / ml to a 50 μM rapamycin solution and mix well; The structural formula of rapamycin is ; The Bacillus thuringiensis mosquito-killing toxin is the Bacillus thuringiensis mosquito-killing toxin Cry4Ba protein; The mosquito-killing activity refers to the mosquito-killing activity against the larvae of Aedes aegypti.

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