Molecular Cloning, Expression And Functional Pre-testing Of BmGMCβ2Gene In Silkworm,Bombyx Mori

The GMC(glucose-methanol-choline) gene family in organisms is a diverse group of flavoenzyme genes, which expressed proteins containing homologous skeleton structure. The proteins are named for the GMC oxidoreductase family. GMC genes were initially found in the microorganisms; and also exist in a large number of insects and other invertebrates. However, these genes are not or rarely found in C. elegans and higher vertebrates. GMC oxidoreductases have different functions, can catalyze different responses. Now, they can be divided into11kinds of functions at least, but there are a trifle functional researches of specific genes in the GMC gene family.Iida’s (2007) research found that, insect genomes containing a large number of GMC genes. In insect, most of the genes in GMC gene family are clustered tandem arrangement in the highly conserved GMC gene cluster, and this gene cluster is included in the one large intron-flotillin-2. Silkworm (Bombyx mori) as a model species of Lepidopteran insects, there are a lot of reports about its genetics, physiology-biochemistry and pathology studies. However, the GMC genes in silkworm are still almost a blank. Our analytic result indicate the silkworm genome contains43GMC family genes, and their distributions in the silkworm genome are consistent with insects in Iida’s study.In this study, we select the BmGMCβ2gene of silkworm GMC gene family to cloning, expressing, and pretesting the function on the basis of the silkworm genome database. Identify the function of the BmGMCβ2gene through the above researches. The main findings are as follows:1. Molecular Cloning and Sequence analysis of BmGMCβ2geneBased on the silkworm genome database, we designed the primers and cloned the target gene BmGMCβ2. Bioinformatics analysis showed that the gene is1875bp in length, encoding a putative protein with624amino acids. The predicting protein molecular weight is69.3kDa and isoelectric point is6.21. It is located on the scaffold scaf3058of16th chromosome in silkworm, containing ADP-bindingβ-α-β fold domain which was shared by GMC gene family. Phylogenetic analysis suggested that the gene is a member ofβ-subfamily in silkworm GMC gene family.2.Expression pattern of BmGMCβ2geneThe expression pattern of BmGMCβ2gene was determined by RT-PCR of different growth periods and different organizations in silkworm. The mRNA transcription of BmGMCβ2gene is mainly concentrated in full appetite of5th instar larvae. At the organizational level, it was expressed in fat body, integument, and trachea, and does not have gender differences.3.The prokaryotic expression and polyclonal antibody preparation of BmGMCβ2geneAfter amplification, we digested BmGMCβ2gene fragment containing specific restriction sites, and subcloned it into the expression vector pET-28a (+). And then the recombinant vector was transformed into E.coli BL21expression strains for inducing expression. By SDS-PAGE electrophoresis detection, the expression product was emerged a band at69kDa; It is consistent with the predicted protein molecular weight, was identified as the target protein. The target protein was purified by His-affinity chromatograph to prepare polyclonal antibody, and then, we analysised the prokaryotic expressed protein by Western blotting. The result showed it appeared a single hybridization signal band about69kDa spot, indicating that the prepared antibodies have good specificity and can be used for further research.4.Tissue localization of BmGMCβ2gene Western blotting assay was performed to detecting the organizational profile of BmGMCβ2gene at the protein level. The result showed that the protein was mainly expressed in the fat body. Further, we detected hybridization signal in fat body by immunohistochemistry, which confirmed the expression of BmGMCβ2protein in fat body of the silkworm.5.Functional pre-testing of BmGMCβ2geneAfter eating the mulberry leaf which smeared with pathogenic microorganism, we detected the expression of BmGMCβ2gene in silkworm, inducing by G-bacteria (S. aureus), the G+bacteria (B. beauveria) and fungi(B. bassiana). The results indicated that the silkworm induced by G+bacteria and fungi, the expression of BmGMCβ2gene was significantly increased; silkworm induced by G’bacteria, the expression of BmGMCβ2gene did not upregulated. The above results revealed that the BmGMCβ2gene may be related to Toll signal pathway of innate immunity in silkworm.