New bacillus strain HSY204 and insecticidal genes and application thereof

A Bacillus, insecticidal gene technology, applied in the application, insecticide, genetic engineering and other directions, can solve the problems of no growth, no report on application, slow growth, etc., and achieve the effect of reducing the risk of large-scale transmission

Active Publication Date: 2021-02-09
HAINAN NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The growth temperature ranges from 20 to 45°C. It grows slowly or does not grow below 10°C. It exists in soil, water, air, and animal intestines. It can produce antibacterial substances, inhib

Method used

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  • New bacillus strain HSY204 and insecticidal genes and application thereof
  • New bacillus strain HSY204 and insecticidal genes and application thereof
  • New bacillus strain HSY204 and insecticidal genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Screening and identification of embodiment 1-bacterial strain HSY204

[0028] (1) Soil sample collection: The sampling site was selected in the tropical rainforest of Hainan Province. In order to increase the probability of obtaining new strains, the sampling site was selected in a place with complete vegetation protection, no human intervention in the ecological environment, and rich soil humus;

[0029] (2) Screening of strain HSY204: Using sodium acetate-temperature screening method, weigh about 5g of soil and put it into 20ml of BPA medium, shake fully, culture on a shaking table at 30°C for 4-5h, bathe in water at 75°C for 15min, take 1ml of supernatant to dilute to 10 -3 、10 -4 and 10 -5 , each pipette 200 μL of NB plates, culture them upside down at 30°C for 3 days, pick single colonies with different shapes and plate them.

[0030] (3) After the bacillus was cultured for 3-5 days to form spores, further use carbofusin staining and optical microscope to isolat...

Embodiment 2

[0031] DNA extraction and insecticidal gene identification of embodiment 2-bacterial strain HSY204

[0032]The genome sequence of strain HSY204 was analyzed molecularly and bioinformatically by using MiSeq Illumina next-generation sequencing and PacBio technology.

[0033] 1. Genomic DNA extraction of strain HSY204

[0034] (1) Inoculate a single colony of HSY204 into about 2-5mL of LB liquid medium, and cultivate overnight at 28°C and 220rpm for activation;

[0035] (2) The next day, transfer 1% to fresh LB liquid medium, and continue to cultivate at 28°C and 220rpm for 4-6 hours until OD600=2.0;

[0036] (3) Collect 1-3mL bacteria by centrifugation at 8000rpm for 2min;

[0037] (4) Wash the precipitate with 1mL J Buffer (0.1M Tris HCl (pH 8.0), 0.1M EDTA (pH 8.0), 0.15M Nacl), centrifuge at 8000rpm for 2min, discard the supernatant;

[0038] (5) Pipette the pellet to resuspend the pellet in 500 μL J Buffer (lysozyme was added to 20 mg / mL before J Buffer is used), incubate...

Embodiment 3

[0048] Embodiment 3-cloning and expression of bacterial strain HSY204 insecticidal gene

[0049] Genomic DNA of strain HSY204 was extracted by alkaline lysis, primers were designed according to the coding sequences of genes orf5878, orf5055, and orf5877, and the genes were inserted into the expression vector pET-30a by means of seamless cloning technology to construct successful recombinant transgenics. into BL(DE3) for inducible expression.

[0050] The SeamLess cloning technology is specifically:

[0051] A. Linearize the vector using double enzyme digestion or PCR amplification

[0052] (1) After enzyme digestion, the linear vector is recovered by gel, and both blunt ends and sticky ends are acceptable;

[0053] (2) PCR amplification to obtain a linear vector, if it is a single band, the PCR product is directly purified, otherwise, it is purified by gel recovery.

[0054] B. Use the designed primers to perform PCR amplification of the target DNA fragment;

[0055] Table...

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Abstract

The invention provides a new bacillus strain HSY204 and insecticidal genes and application thereof. The new bacillus strain HSY204 is classified and named as bacillus cereus HSY204, and comprises three novel insecticidal genes. It is indicated through bioactivity assay that the strain HSY204 and the insecticidal genes thereof have very high toxicity to aedes aegypti larvae, have obvious influenceson the growth of the mosquitoes, can be applied to prevention and control of the mosquitoes, and effectively reduce the risk of large-scale propagation of mosquito-borne diseases.

Description

technical field [0001] The invention relates to the field of environmental microbes and the technical field of molecular biology, in particular to a new bacillus strain HSY204 and its insecticidal gene and application. Background technique [0002] Mosquito is a kind of pest that we are familiar with, and it is of a great variety, and there are more than 3200 known in the whole world, and wherein the known mosquito class of our country reaches 18 genera, 48 subgenuses and 371 kinds or subspecies. Mosquitoes not only seriously affect our lives, but more importantly, they are the vectors of spreading various diseases. There are more than 10 kinds of mosquitoes that suck blood and can transmit diseases in my country at present. Diseases transmitted by mosquitoes occupy an important position in preventive medicine, because mosquitoes have strong transmission power, high incidence and great harm. The diseases it spreads mainly include malaria, dengue fever, filariasis, Japanese...

Claims

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Application Information

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IPC IPC(8): C12N1/20C07K14/32C12N15/31C12N15/10A01N63/22A01P7/04C12R1/085
CPCC07K14/32A01N63/22C12R2001/085C12N1/205Y02A50/30
Inventor 张文飞吴江玉贾璐羽何佳利范子钰蔡雨辰张旭东王锐萍
Owner HAINAN NORMAL UNIVERSITY
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