The Preliminary Research Of Laccase Gene As A New Target Of Cotton Pest Control

Laccases have three Cu-oxidase domains in polyphenol oxidase, which catalyze theoxidation of a vast array of aromatic substrates along with the reduction of molecular oxygento water. The mechanism of catalytic reaction is a process of oxidation and reduction byelectron transfer. It has been reported that laccases play an important role in cuticle tanning(or sclerotization and pigmentation) for insects growth. The enzymes can be used for manyindustrial, environmental, diagnostic, and synthetic uses. The results are as follows.(1) With the specific primers we amplified the target fragment of the laccase gene inHelicoverpa armigera by RT-PCR method. The length of cDNAs is2211bp, encoding737amino acid, and contains an open reading frame (ORF). Analysis of the sequence of laccasegene by bioinformatics shows there is a high homology between cotton bollworm and otherinsects. Detailed analysis and prediction of the structure of protein were also made.(2) Through the cotton bollworm laccase gene sequences, the sequences of laccase,bioinformatics analysis for the current study of bollworm laccase gene provides a scientificbasis. Through analyzing the sequencing results of bioinformatics, cotton bollworm laccasegenes with other lepidoptera insect laccase has higher homology, and the protein structure oflaccase to make detailed analysis and prediction. The alpha helix in the secondary structure ofCotton bollworm laccase is occupied1.22%, beta folding accounted for22.93%, ring andother random structure about75.85%.(3) This experiment tested the directional cloning method. By Xcm Ⅰ digestion ofpET17ba, linear directional cloning vector a T or C at respective3’end. Analysis show thatthis method can ensure the orientation of the PCR products. PCR amplification of laccasegene for test prokaryotic expression vector of laccase’s construction is successful. The newcloning method will improve the efficiency of vector construction, which can reduce the costof experiment. Sequencing analysis shows that Laccase expression vector constructed by theabove method is correct.