The Bioassay And Toxic Constituents Of A.Cantonensis With Bacillus Thuringiensis Isolated From Lichens

With heat treatment and best selective culture medium, 6 novel B. thuringiensis strains had been isolated from 48 samples of lichens,which was a kind of non-vascular plant. They were named from BRC—ZQL1 to BRC—ZQL6.Physiological and biochemical determination was executed to the 6 novel strains for understanding their biological characteristics. The results showed that :6 strains were both different. The PCR-RFLR technique was applied in detecting the contents of the isolated Bt strains. The results showed that 5 classes of cry1 gene existed in all of the 6 strains, which were cry1Ad, cry1Ba, cry1Cb, cry1Da and cry1Ea, and cry1Ia1 and cry1Ib1), cry2 gene existed in 5 of them, and a total of 2 classes(cry2Ah and cry2Ah) . These strains also contained cry9 and cry1I genes. Some special DNA fragments detected from BRC-ZQL5,BRC-ZQL6 indicated the existence of new genes.By different concentrations of the bacterial liquid of BRC-ZQL1 to BRC-ZQL6 as well as 3 Bt strains saved in Laboratory (Bt 8010, Bt HD1, Bt 010), preliminary identification of insecticidal activity on A.cantonensis was carried out, and great difference was detected. In the tested strains, BRC-ZQL3 was the best toxic, the LC50 of which were 2.85415mg/mL( 24 h) and 1.10599 mg/mL(48 h), respectively. It demonstrated that the parasporal crystals of BRC-ZQL3 had significant toxic effect on A.cantonensis.Using the total DNA of BRC-ZQL3 as template, the cry2 gene was detected. Combining the PCR products with the cloning vector and transforming the recombinant plasmids into JM109 competent cells, the target gene ORF was obtained, which sequenced with 1899 bp. It was submitted NCBI with the GenBank Accession No.: FJ550343.1, and was named cry2Ah2 gene as a novel gene by International Bt Naming and Classification Committee. Analysis by Bioinformatics software showed that the gene conduced to a translation of a protein with 632 amino acids, and the predicted molecular weight was approximately 70.84 kDa. Homology alignment of the Protein and analysis of physical and chemical properties were done and the two-dimensional structure and three-dimensional structure were also forecasted.Transforming the constructed expression vector pGEX-cry2Ah2 into E. coli BL21 (DE3) provided us with the recombinant plasmid pGEX-cry2Ah2 in E. coli BL21 (DE3). SDS-PAGE resulted that cry2Ah2 gene co express the recombinant strain in the recombinant E. coli successfully.Inducting the recombinant E. coli with IPTG, the bacterial lysate is used for the original toxicity test for A.cantonensis. The corrected mortality of 24 h and 48 h was 12.77% and 22.91%, respectively. It was showed that the lysate had nematicidal toxicity for A.cantonensis in some degree, and Cry2Ah2 protein had virulence factors for the toxicity.In order to improve the spore yield of the Bt strain BRC-ZQL3, the mathematical statistics method was employed to optimize the fermentation medium for spore production of BRC-ZQL3. Firstly, the mono-factor experiment method was adopted to determine the effects of different carbon and nitrogen sources, initial pH and other factors on sporulation of BRC-ZQL3. Secondly, the three important factors, such as CaCO3, corn meal and yeast extract, were found after the screening of eight factors by applying the Plackett–Burman experimental design at two levels. Identified by response surface methodology wtih the three factors at three levels, the optimal medium compositions was as follows: 2 % Soybean powder,0.42 % corn meal,0.39 % yeast extract,0.025 % KH2PO4,0.035 % MgSO4.7H2O,0.04 % CaCO3 with the initial pH 7.2. After optimizated, its spore production was raised to 3.92×10~8/mL and the number increased by 60.66 %, comparing with the initial fermentation medium. Between the actual value and the predictive value of response surface model, the error was only 2.9 %.In this study, Bt is firstly isolated from lichens, such as special habitats plants. It can not only enhance the understanding of the living environment of the Bt strains, but also expand new sources to the collection for new wild-type Bt strains. Meanwhile, it is also first to use Bt toxicity test on A. cantonensis. Filtered a control role of A.cantonensis in the Bt strains, the Bt strain with strong insecticidal activity on A. cantonensis was found. and their virulence factors were studied, it is a useful exploration to to find new ways to control A.cantonensis.