Cloning And Expression Of Chitinase Genes From Locusta Migratoria Manilensi And Metarhizium Anisopliae Var. Acridum

The cuticle and the peritrophic matrix of insects acted as physical barriers to pathogens and environmental hazards. Both were composed primarily of chitin and protein. The degradation of chitin by chitinase was vital step in the life cycle of insects because insects undergo periodic ecdysis and metamorphosis. The old layers of the cuticular exoskeleton should be digested prior to ecdysis and metamorphosis. During the molting process, chitin in insect cuticle was degraded by chitinase. And penetrating host's cuticle is one of the key steps in fungi infection, too. Several chitinase genes have been isolated and integrated into transgenic plants to enhance their defence mechanisms, and inserted into insect pathogens to increase their deleterious effects on insects. So, research on chitinase genes might provide an important clue for the utilization of these enzymes as a biocontrol agent of insect pests.This research amplified chitinase genes by PCR from Locusta migratoria manilensi and Metarhizium anisopliae var. acridum. We constructed the yeast expression vectors. The expression of chitinase in Pichia.pasteoris was studied. The main achievements were as follow:â‘ Cloning and sequence analysis of chitinase gene from Locusta migratoria manilensi midgutThe full cDNA (GenBank accession no: EF092841) of Locusta migratoria manilensi chitinase (LmChi) was cloned by means of 3'-RACE and 5'-RACE. The results showed that the cDNA was 1 604 bp in length and contained an open reading frame (ORF) of 1 452 bp, which encoded 483 amino acids. The deduced amino acid sequence, which composed of a signal peptide, a chitinase active site, a C-terminal Serine-rich region and a Chitin-binding domain normal present in chitinase, showed a high similarity with other insect chitinases from family 18.â‘¡Tissue expression of LmChi geneIn this study, we determined tissue expression of LmChi gene by Semi-quantitative RT-PCR. The results indicated that LmChi expressed only in midgut of L. migratoria manilensis, but not in integument, foregut and hindgut.â‘¢Cloning and sequence analysis of chitinase gene from Metarhizium anisopliae var. acridumThe full cDNA and DNA of Metarhizium anisopliae var. acridum chitinase (MaChi) was cloned by means of RACE and library screen. Some sequences were submitted to GenBank(GenBank accession no: DQ097518). The results showed that the DNA was 1 973 bp in length and contained 3 introns. The cDNA was 1 483 bp in lenth and contained an open reading frame (ORF) of 1 27 bp, which encoded 424 amino acids. Like LmChi, the deduced amino acid sequence, which composed of a signal peptide and a chitinase active site normal present in chitinase, showed a high similarity with other chitinases from family 18. But the sequence lacked Chitin-binding domain.â‘£Expression of LmChi and MaChi in Pichia.pasteorisThe cDNA encode mature peptide of LmChi and MaChi were inserted into pPIC9K. The recombinant of pPIC9K-LmChi and pPIC9K-MaChi plasmids were successfully transforned into Pichia pastoris KM71 by electrotransformation. In shake-flask culture induced by methonal, the chitinase activity in surpernatant was detected.