Cloning And Characterization Of Insecticidal Crystal Protein Gene And Chitinase Gene From Bacillus Thurinriensis

Bacillus thurinriensis is one of the most important insecticidal microbes in the field of biological control whose remarkable characterization is that it can produce parasporal crystals during the formation of spore. Parasporal crystals are usually called insecticidal crystal proteins (ICPs) or 8-endotoxins. ICPs are not pathogenic to beneficial insects and mammals, but they are very specific to the invertebrate pests against which they have activity. More than 200 ICP genes have been cloned till now, but some destructive major pests are becoming more and more resistant to the Bt-lCPs. Therefore, it is imperative to exploit new resources of Bt and clone or reconstruct new genes.Chitin, a B-1,4-linked homopolymer of N-acety-D-glucosamine, occurs in insects as a major component of the cuticle and of the peritrophic membrane, a protective sleeve lining the gut of many insects. Chitinase can catalyze the conversion of insoluble chitin to its monomeric components. It is found that chitinase can also increase the insecticidal activity of Bt, which can overcome or delay the resistance of insect to Bt.In this dissertation, two pairs of primers were designed. The insecticidal crystal protein cry 1Aa13 gene was cloned from large plasmids of Bacllus thuringiensis subsp sotto, and the chitinase ichi gene was cloned from chromosomal DNA of Bacllus thuringiensis subsp israelensis. The nucleotide sequences of the two genes have been determined; the sequence analysis and functional prediction have also been done. The main results are presented as the following.1. Setting up the miniscale preparation method for large plasmids DNA horn Bacillus thuringiensis. The plasmid was purified using PEG 6000 instead of phenol and chloroform. Experiments demonstratethat consequences from this protocol were stable, the quality and yield of plasmid DNA produced from it have met the standard for many molecular biology experiments.2. Employing computer analysis of cry1A gene sequences, a pair of specific primers were designed. With these primers, PCR was performed and an insecticidal crystal protein gene (about 3.6kb) was obtained from Bacillus thuringiensis subsp sotto. The sequence was registered in GenBank (Accession Number is AF510713), which was designated crylAa13 gene as a novel gene by Bt o-endotoxin nomenclature committee.3. cry1Aal3 gene sequence includes an open reading frame (ORF) of 3S43bp, which contains 1180 amino acids with a predicted molecular mass of 133.50kDa. The amino acid sequence of crylAa13 shows more than 95% identity to that of crylAa. It contains three conserved domains, viz. domain I, domain II, and domainin.4. Employing computer analysis of chitinase gene sequences, a pair of specific primers was designed. With these primers, PCR was performed and chitinase ichi gene was obtained from Bacilku thuringiensis subsp israelensis. The sequence was registered in GenBank (Accession Number is AF526379).5. ichi ene sequence with length of 2570bp is a complete gene comprised of a promoter, coding regions and a terminator sequence. The 2067bp open reading frame includes 688 amino acids with a predicted molecular mass of 75.79kDa and an isoelectric point of 5.90. Ichi contains a signal peptide (46AA) and three functional domains, viz. a catalytic domain (105AA), a chitin-binding domain (40AA), and a fibronectin type III-like domain (74AA).