Heterologous Expression Of Chitin Deacetylase Of Locusta Migratoria And Its Function In The Locust Hindgut

The locusts are important agricultural pests in China.At present,the prevention and control mainly rely on chemical pesticide,however,unreasonable chemical control not only causes environmental pollution,but also leads to resistance of locusts and harm to non target organisms.Therefore,to explore a new,environmentally friendly locusts prevention methods are becoming more and more important.The development of insects periodically takes place the old epidermis in the process of chitin metabolism.Chitin is a kind of unique component of the epidermis,digestive tract and body tissues.In this,chitin deacetylase(CDAs)plays an important role in the process of chitin metabolism,which can catalyze the hydrolysis of acetyl group of N-acetyl glucosamine in chitin,that composed by ?-1,4 glycosidic bonds,transform chitin into deacetylation of chitin(chitosan).Therefore,research and development of environmentally friendly pesticides based on insect chitin degradation pathway receives much concern.In this study,we put the LmCDAs gene as the research object.Firstly,we used eukaryotic expression system,expressed the target protein gene in vitro,purified target protein,and detected their enzymes activity,thereby to explore the function of the protein genes in vitro;Secondly,We also used prokaryotic expression system,expressed the target protein genes in vitro,purified target proteins,and preparation the specific polyclonal antibodies of LmCDA1 and LmCDA2,which provides a basic material to explore the functions of LmCDA1 and LmCDA2 gene in insects body.Finally,based on the research foundation of the research group,we used specific preparation of polyclonal antibody of LmCDA1 and LmCDA2 to explore the effects of LmCDA1 and LmCDA2 gene on the development of the locust hindgut.This study provides an important basis for the study of the functional properties of genes in chitin metabolism.This paper is divided into the following three parts:1.Study on eukaryotic expression,affinity purification and enzyme activity of chitin deacetylase in Locusta migratoriaUsing PCR method to amplification LmCDA1,LmCDA2 a and LmCDA2 b gene of Locusta migratoria,after purification and recoveried,detected the recovery products by agarose gel electrophoresis.Choosed pFastBac?-Dual as the intermediate vector,then using double enzymes digested the target gene sequence and pFastBac?-Dual intermediate vector,construction the recombinant plasmids,and then constructed the recombinant baculovirus.Recombinant baculovirus was transfected into Sf9 cells,getted the recombinant protein of LmCDAs,the expression of recombinant protein was confirmed by western blot and purification of recombinant protein by 12% SDS-PAGE gel electrophoresis,and preliminary enzyme activity assay.Conclusion: LmCDA1,LmCDA2 a and LmCDA2 b had deacetylation function in vitro of Locusta migratoria,and their activity showed significant difference.2.Prokaryotic expression,purification and antibody preparation of LmCDA1 and LmCDA2 in Locusta migratoriaPrimers were designed to clone and purify the target genes,p ET32 a was selected as the intermediate vector to construct recombinant plasmids pET32a-LmCDA1 and pET32a-LmCDA2,the recombinant plasmids were constructed and transferred into E.coli to induce the expression of the target protein,the expression products were detected by western blot technique.The expressed proteins were purified by Ni-NTA affinity chromatography column,the purified components were detected by 12%SDS-PAGE method,then the proteins concentration were detected by BCA method,when the concentration run up to 10 mg were sent to the biological company to prepare the antibody.Antibodys synthesis were performed using the western blot technique for antibody validation.Conclusion: Recombinant plasmids pET32a-LmCDAs could be induced the expression of the target protein in E.coli by 100 mM IPTG,whichinduced 4h at 37?.Moreover,a large number of target proteins could be obtained by using the prokaryotic expression system of E.coli,which achieved the required concentration and purity of antibody preparation.3.Localization of Lm CDA1 and Lm CDA2 and functional analysis in the hindgut of Locusta migratoriaThe localization of LmCDAs gene in the locust hindgut were detected by immunofluorescence.To investigate the effect of LmCDAs gene on the locust hindgut development by means of RNAi interference and transmission electron microscopy.Conclusion: Immunofluorescence detection results showed that LmCDA1 and LmCDA2 were located in locust hindgut cytoplasm at day8 of the fifth-instar nymphs,and RNAi and TEM results showed that the LmCDA1 and LmCDA2 gene plays a key role in chitin layers structure of locust hindgut.