Cloning And Eukaryotic Expression Of Uridine Diphosphate-Glucosyltransferase Gene BmUGT4965 In The Silkworm, Bombyx Mori

Glycosyltransferases (GTs) are ubiquitous in animals,plantss,bacteria and viruses as a large class of enzymes in nature. They catalyze the transfer of the sugar residue of an active sugar donor (uridine diphosphate-sugar as usual, UDP-sugar) to an acceptor molecule (aglycone) completing the glycosylation reaction. Glycosylation changes the property of aglycone such as bioactivity,solubility,and the transport characteristics between cells, exerting diverse functions in organisms. The donor include UDP-glucuronic acid,UDP-glucose,UDP-galactose,UDP-xylose and so on. While the acceptor molecules include various kinds of endogenous and exogenous compounds. Mammalian uridine diphosphate glycosyltransferase (UGTs) use UDP-glucuronic acid as sugar donor, catalyze the glucuronidation of many endogenous substrates such as bile acids,steroids and so on, participating in their metabolism and balance regulation. In addition, they also catalyze the glucuronidation of exogenous compounds such as carcinogen,toxic substance,and therapeutic drugs, playing a centra part in the detoxication and drugs clearance. The UGTs in plants mainly use UDP-glucose as sugar donor, catalyze the glucosylation endogenous substrates such as plants and secondary metabolite, playing an important role in the physiological processes such as the regulation of plant growth and the protection against pressure. Moreover, they also take affect in the detoxication of herbicides as xenobiotics.The UGTs in mammalian especially in humans have derived extensive and deep research when their potential value in pharmacectic and clinic attract researcher because they participae in detoxication of carcinogen and clearance of therapeutic drugs. There are also many reports about the UGTs in plants, while those in insect is very few. The present results indicate that the UGTs found in insect and the related ones found in mammalian share many similar characteristics, and play equally important roles. Silkworm has been devolped to be a model organism to research lepidoptera insects. Multiple genes of UGTs in silkworm have been identified, the research of them may make for understanding their functions in the growth and development of silkworm, but also provide molecular basis for research of UGTs in other species. Our research cloned a UGTs gene BmUGT4965 in silkworm. We analyzed the sequence characteristics and the expression trait in each tissue of silkworm on the 3rd day of fifth-instar larvae, made an eukaryotic expression of the gene and detected its catalysis activity to tested substrates. The main results as follows:1 The clone and sequence analysis of BmUGT4965 geneWe have cloned the Bm UGT4965 gene (Access No. EU873318). The gene is located on the 25th chromosome and complete CDS is 1578bp. The sequence comprises four exon and three introns and all exon/intron boundary are consistent with typical GT-AG rule. The protein sequence encoded by Bm UGT4965 gene contains the typical UDPGT domain, falling into two main domains as the C terminal and N terminal. Comparison with other sequences indicates that the C terminal half are highly conserved, while the N terminal half which is to bond aglycone with diverse structures share little identity. Similar to the UGTs in mammalian, BmUGT4965 protein is located in endoplasmic reticulum as a membrane-bound enzyme. The N terminal contains a putative signal sequence, immediately after which is a conserved region. In the mammalian UGTs, this region has been identified as an oligomerization domain. In addition, there is a N-glycosylation site in the Nterminal sequence. Thus, the protein is oligomerization and glycoprotein in nature. There is a hydrophobic transmembrane domain and immediately followed by a basic motif, this two region affect in common to anchor the protein in the endoplasmic reticulum with the N terminal protruding into the lumen2 The expression of BmUGT4965 gene in different silkworm tissuesWe detected the expression of BmUGT4965 gene in midgut,hemolymph,silkgland,int egumentum,head,fat body,vasa malpighii,testis and ovary all together nine tissues of sil kworm on the 3rd day of fifth-instar larvae with RT-PCR technology. The result shows that it express highly in silk gland and fat body, and weakly in midgut and vasa mucosa. The tissue specific is fairly high.3 Eukaryotic expression of BmUGT4965 geneWe constructed the express recombinant vector and expressed the recombinant fusion protein in Hi-5 cell line with Bac to Bac baculovirus expression system. Detected with SDS-PAGE and Western blotting, the interest protein is approximately 64.5kDa.4 The activity detection of BmUGT4965 proteinDetecting the crude catalysis activity of recombinant protein with quercetin and kaempferol as substrates, the result demonstrated the protein can catalyze the two substrates, generating one and two production respectively.The dietary flavonoids absorbed by silkworm from mulberry leaf is glucosylated at the 5-O position in the midgut as the first-pass metabolite and subsequently glucosylated at the 4' -O position when the first-pass metabolite get into the silk gland or fat body via the hemolymph. The BmUGT4965 protein may participate in the latter metabolism, resulting the exerting of anti-oxidant function by flavonoids in silkworm cocoon.