Cloning And Expression Analysis Of The CDNA Encoding Amide Glucose Transferase In Silkworm,Bombyx Mori.

Glucose transferase(GlcNAcase)is a group enzymes can catalyze N-alkylation of sugar chain generation,which was mainly distributed in the Golgi apparatus and Endoplasmic reticulum of biological cell.Various types of carbohydrate and other non-carbohydrate compounds can be modified and the biological function of various molecules can be achieved by GlcNAcases.GlcNAcases of insects play a very important role in a variety of physiological and biochemical processes,including exogenous and endogenous cellular substrates synthesis,decomposition,processing,modification and other aspects.The gene cDNA which encoded GlcNAcase in silkworm was cloned and expressed in this research.The main results are as follows:1.The specific primers were designed based on the information from the GenBank and other related information of the known sequences to clone the GlcNAcase cDNA.The GlcNAcase cDNA sequence was 1113 bp,which encoded 370 amino acid peptide including a catalytic sequence contains 45 amino acids domain.Phylogenetic tree suggested that the distance between the silkworm B.mori and Coleoptera was the nearest,and that GlcNAcases in B.mori and other insects belong to the glycosyl transfer enzyme gene family,which was named as BmGlcNAcaseA.2.In this research,BmGlcNAcase A sequence was inserted into the pET-28A(+)vector.Recombinant plasmids were transfected into E.coli Transetta(DE3),and the protein of BmGlcNAcaseA was induced with IPTG in the E.coli.The results showed that the target protein was correctly expressed through the analysis of SDS-PAGE and Western Blot.The recombinant protein was purified with the method of NI-NTA affinity chromatography column and polyclonal antibody was prepared.The titer of antibody was 11400.3.The expression level of BmGlcNAcaseA in the ovary and other tissues of the fifth instar in B.mori was analyzed by the method of SDS-PAGE and Western Blot.The results showed that the expression of BmGlcNAcase A was the highest in ovary and epidermal,the next is in the midgut and fat body,which blood?testis and malpighian tube was the least.4.Fifth-instar larvae of B.mori were injected with Nuclear polyhedrosis viruses,Micrococcus luteus,Escherichia coli or Beauveria bassiana.The results suggested that the BmGlcNAcaseA may be involved in the process of immune response induced by exogenous microorganisms.The expression level of BmGlcNAcaseA gene was firstdown-regulated and then up-regulated,indicating that the gene on the immune response of different microorganisms is not the same as.The analysis showed that the BmGlcNAcaseA is relatively sensitive to NPV,and response slowly to other bacteria.The expression level of BmGlcNAcaseA reached the maximum at 6 h in B.mori induced with Nuclear polyhedrosis viruses,however the peak reached the maximum at 9 h induced with other bacteria,and the expression level reached the lowest at 12 h.In summary,the expression level of BmGlcNAcaseA in different tissues is different,which is closely related to its function in the organization B.mori,and the clone and expression of BmGlcNAcaseA can provide a foundation for the further research of its function in B.mori and other insects.