Cloning, Sequence Analysis And Eukaryotic Expression Of Chitinase Gene(Group Ⅰ) From Lymantria Dispar

Chitin, a linear polysaccharide of N-acetyl-D-glucose amine polymerization, exists in nature in various forms and is widely distributed in bacteria, fungi, algae, arthropod and so on. Chitin which plays an important role to support the body and protects the insects against external damage is a major structural component of body wall, peritrophic membrane, trachea, and adipose tissue of insects. Insect chitinase is a key biological enzyme in the decomposition of chitin metabolism and is indispensable throughout the process of growth and development of the insect. It is necessary to express a certain amount of chitinase in the process of digestion, metamorphosis, infection of insect. In view of the insect chitinase s importance, studying insect chitinase gene and protein characteristics provides the basis for the biological control of insect pests and applying its gene on the recombinant baculovirus which is a significant try to explore new methods of prevention and treatment of plant diseases and insect pests.Forest pest Lymantria disparand were fed by artificial methods. First-strand cDNA was synthesized from total RNA and Lymantria disparand chitinase gene was cloned with the method of RACE. The amino acid sequence inferred from the above chitinase gene is analysed by means of related software and online servers to obtain bioinformatic. Bac-to-Bac eukaryotic expression system was built to express Lymantria dispar chitinase and to obtain recombinant viruses which were largely amplified in Helicoverpa armigera larvae.The cloned Lymantria dispar chitinase gene is1895bp in length and contains an open reading frame of1737bp, encoding a polypeptide of578amino acid residues with a predicted molecular weight of64.4237kDa and a pI of5.50. The chitinase gene shows the most highly homology with Phyllonorycter ringoniella and Helicoverpa armigera that belong to Lepidoptera. The hydrophilic polypeptide with5phosphorylation and38glycosylation modification sites has a predicted signal peptide with21amino acid residues but has not transmembrane structure. Phylogenetic tree of the30species of insects including Lymantria dispar according to their chitinase gene was built to study its evolution. Active proteins with a molecular weight of66kDa obtained in Sf9cells were purified by the Ni column. Recombinant virus with chitinase gene was successfully amplified in Helicoverpa armigera.It is the first time to report research on Lymantria dispar chitinase on molecular level in this paper. Investigations on bioinformatical analysis and eukaryotic expression of Lymantria dispar chitinase have contributed to making better understanding on function, properties and activity of insect chitinase. Accomplishment of the subject offers theoretical data for in-depth understanding of the insect chitinase and provides an important basis for the next step of the direct application of the Lymantria dispar chitinase or application of this gene in transgenic, and also affords a theoretical reference for exploring new methods of biological control of Lymantria dispar.