Study On Genes Expression And Function Of UDP-glucosyltransferases In The Silkworm, Bombyx Mori

The domesticated silkworm, Bombyx mori serves as a model for Lepidoptera. The processes of pigment absorption, modification and transportation are under control of complex gene regulations. In Bombyx mori, flavonoid compounds have been found as major pigments in the green cocoon shells, while the formation of glucoside is the predominant pathway for biotransformation of flavonoids from mulberry leaf. Mechanisms surrounding cocoon coloration regulation in Bombyx mori have become a main focus in animal studies for regulation of pigment absorption.UDP-glucuronosyltransferase (UGTs) is a multigene family and occurs in animals, plants, insects, and microbes. These enzymes catalyse the glucuronidation of exogenous compounds such as dietary constituents, drugs, and environmental intoxicants, as well as endogenous compounds such as steroids, other hormones, neurotransmitters, and bile acid. UGTs, which are membrane-bound protein, are located in the lumen of endoplasmic reticulum. In the silkworm Bombyx mori, over 42 UDP-glucosyltransferase (UGTs) genes have been revealed by recent determination of the complete genome sequence. In addition, glucosidation in insects is known to be involved in in several processes, including cuticle formation, pigmentation and olfaction, although the processes were validated only at the biochemical level in a few cases.Flavonoids are the major constituent of pigments that have been characterized to be involved in green cocoon coloration of Bombyx mori. Recently, metabolic conversion processes of flavonoids and their glucosides by silkworm have been postulated. Flavonoids are absorbed from mulberry leaves, transferred from the midgut to the silk gland via the hemolymph, and they are accumulated in the layers of the cocoon sericine. A class of UDP-glycosyltransferases (UGTs) is involved in this process. The formation of glucoside is the predominant pathway for dietary flavonoids. Studies on UDP-glycosyltransferase activity in insects have been mainly focused on only limited information. It has been reported that quercetin 5-O-glucoside was the predominant metabolite in the midgut tissue, while quercetin 5,4'-di-O-glucoside was the main constituent in the haemocyte and silk gland. Dietary flavonoids are metabolized and accumulated in cocoons, as a result of leading to green coloration. Recently, identified Gb locus, a an independent loci is suggested to be associated with the green cocoon trait, encodes a quercetin 5-O-glucosyltransferase that catalyzes the regioselective formation of quercetin 5-O-glucosides. Therefore, we speculate that the glucuronidation of flavonoids is a key factor to green cocoon coloration.1. Firstly, we performed RACE experiments and determined the full lengthsequences in Silkworm strains p50T. This cDNA sequence has been deposited in GenBank under accession HQ833816. We employed RT-PCR to detect and quantify the expression levels of UGT30 genes during differently developmental stages and tissue distributions of fifth instar day 3 larvae of the p50T strain.2. For investigating the linkage between the cocoon pigmentation and UGT genes expression, we chose seven UGT genes of UGT013859(UGT59), UGT013830(UGT30), UGT013860(UGT60), UGT013834(UGT34), UGT010286(UGT86), UGT010289(UGT89) and UGT013865(UGT65) which were expressed in silk gland or in midgut of Dazao (P50). The qualitative and quantitative expression analysis of these genes were confirmed by RT-PCR, which were expressed in silk gland, midgut and gonad from varieties of C108 and HY with white cocoon, P50 and G1 with green cocoon, Y12 with yellow cocoon and sex-limited yellow cocoon race of Ys. UGT86,UGT89 and UGT30 were highly expressed highly expressed in silk gland and midgut of green cocoon varieties but lowly in white ones. Thus, these genes are most likely to be involved in pigment transport. This study also demonstrated the existence of some differences in mRNA of varieties with different cocoon colors.3. To evaluate the functional role of UGT30 in cocoon pigmentation, we used RNA interference by injecting UGT30 dsRNA to suppress the production of UGT30 in the Race of p50T. In order to knock down expression of UGT30 in the whole period of fifth instar larvae , 5 ug/insect was injected into the hemolymph twice in the day 1 fifth and day 4 fifth instar larvae using a micro-injector. We found that silkworm prepupae are highly sensitive when they were injected with UGT30 dsRNA. Particularly, after we injected UGT30 dsRNA, their bodies were growing slowly and their pupation rates dramatically decreased.4. UGT30 and UGT86 varied with the varieties of different cocoon colors, showing a typically specific expression pattern of cocoon color. To confirm this further, we examined in the biocatalytic synthesis of glycosides with potential medicinal properties using recombinant UGT30 and UGT86 expressed then. We expressed recombinant? recombinant UGTs in Sf9 cells using Bac-To-Bac baculovirus expression system. In the present study, quercetin was investigated for glucuronidation reactivities with recombinant UGTs. HPLC analysis also detected a second product was formed with recombinant UGT86, and the blank Sf9 cells as control.