Cloning And Expression Of Collagenase Gene ColB1 From Bacillus Thuringiensis And Its Function In Insect Infection

Bacillus thuringiensis (Bt) is a worldwide used pesticide and plays an important role for controlling many kinds of harmful insects. As an important pathogen of insects, Bt produces a variety of virulence factors allowing the bacteria to invade the host, and to resist and to growth in this hostile environment, such as degradative enzymes and toxins, including Phospholipases C, Hemolysins, Proteases, Collagenases etc. In this thesis, we studied the function of Bacillus thuringiensis collagenase Co1B1 in insect infection.1. Cloning and expression of colBl gene and its activity detection.Collagenase gene colBl was predicted and cloned from Bacillus thuringiensis strain YBT-1520. The total length of colBl gene was 2898 bp, which encoded 965 amino acids with predicted molecular weight 110 kDa. The colBl gene was cloned into vector pET28a and expressed in E.coli. The purified protein Co1B1 could produce hydrolysis cycle on gelatin-agar plate, and when using type I-collagen as substrate, the enzyme activity of Co1B1 protein was determined as 27.2 U/mL, and the specific activity was 2709 U/mg by the ninhydrin method.2. Co1B1 could enhance the insecticidal activity of Cry1Ac against Helicoverpa armigera.The Diet incorporation method and the Surface coating method were used for determining the synergistim of Co1B1 protein and Cry 1 Ac protein against Helicoverpa armigera larvae. The bioassays result obtained with Diet incorporation method indicated that high concentration Co1B1 protein could enhance the toxicity of Cry 1 Ac. While with Surface coating method, it was found that the mortality of all of the group adding Co1B1 protein were higher than or equal to the group of Cry1Ac protein alone, and the synergistim was more obvious.3. The function study of BMB171 Co1B1 protein in insect infection.Sequence search in Bacillus thuringiensis acrystalliferous stain BMB171 revealed a protein BMB171_C0475 showing 99% identity at amino acids sequence level to Co1B1 from YBT-1520. First of all, we proved that colBl gene could transcript and express in BMB171 with RT-PCR and Green Fluorescent Protein transcriptional fusion method, and then the colBl gene was knocked out in BMB171 with homologous recombination. The bioassays against Helicoverpa armigera were carried out by mixing the mutant spores and Cry 1 Ac protein quantitatively. The result showed the toxicity of mutant spores mixed with Cry 1 Ac protein was lower than that of the wide-type strain, but there was no significant difference. After cry1Ac gene was transferred into mutant BMB1242 and wide-type strain BMB171 respectively, the bioassays were then performed using fermentation broth directly. The mortality caused by mutant BMB1242-1 Ac was distinctly lower than that of the control strain BMB304-lAc, which demonstrated that deletion of colBl reduced the virulence of Bacillus thuringiensis against Helicoverpa armigera.The second part in this thesis, a Bacillus thuringiensis expression vector named pBMB1A was constructed by cloning BtⅠ-BtⅡpromoter, SD-sequence, and transcription terminator of crylAc gene, and adding His-tag and the following multiple cloning sites (MCS) from pET28a into the shuttle vector pHT304. CrylAc and other five cry genes with atypical BtⅠ-BtⅡpromoter including cry2Ab, cry5Ba, cry6Aa, cry7Ba, cry55Aa were cloned into pBMBIA. Microscopic observation showed that the recombinant strains containing crylAc, cry5Ba, cry7Ba and cry55Aa could form parasporal inclusion bodies, and all of them could produce the predicted crystal proteins bands when detected with SDS-PAGE. Meanwhile, we selected crylAc to determine the effect of His-tag on its insecticidal activity, and the bioassays showed that there was no significant difference between the LC50 value of the recombinant strain and the control strain. The expression vector we constructed was suited for rapid cloning and expression of different cry genes. Also another expression vector named pBMBC2 was constructed by cloning ctc2 gene promoter. The cry1Ac gene was cloned into pBMB1A, and the result showed that the recombinant strain could produce the crystal protein bands and form parasporal inclusion bodies.