All organisms live in an environment that contains natural and man-made potentially harmful chemicals. Extensive studies of detoxification in the vertebrate liver provide a framework to the study of detoxification mechanisms in other systems 123. Several biotransformation enzymes participate in the detoxification process, they are cytochrome P450 superfamily 45, short chain dehydrogenase/reductase (SDR)1 family 6, glutathione S-transferases 7 and UDP-glucuronosyltransferases 2.

Biotransformation enzymes related to those found in vertebrates have also been found in insects and are likely to play equally important roles. Cytochrome P450s and glutathione S-transferases in particular have been implicated in insect resistance to pesticides 8. UDP-glucuronosyltransferases are part of a superfamily of UDP-glycosyltransferases (UGTs, EC2.4.1.-) that are found in all living organisms, including animals, plants, bacteria and viruses, suggesting an ancient origin 91011. UGTs are a superfamily of enzymes that mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones), thus regulating properties of the acceptors such as their bioactivity, solubility and transport within the cell and throughout the organism. The UDP-sugar may be UDP-glucuronic acid, UDP-galactose, UDP-glucose, or UDP-xylose.

The mammalian UGTs using UDP-glucuronic acid as a glycosyl donor have attracted considerable attention in pharmaceutical and clinical research due to their central role in the detoxification of foreign chemicals such as carcinogens and hydrophobic drugs. These enzymes are located in the lumen of the endoplasmic reticulum and are membrane-bound protein. In addition to the detoxication of exogenous substrates, they are involved in a range of other physiological processes, including olfaction and metabolism of bile acids and steroids 12.

Like plant UGTs, insect UGT enzymes also use UDP-glucose rather than UDP-glucuronic acid as sugar donor 131415. Also, similar to the vertebrates, both endogenous and exogenous substrates are subject to glucosidation in insects. UGT activity on endogenous and exogenous compounds has been reported in a range of insect species 16. Insect UGTs play an important role in detoxication of plant allelochemicals encountered by many insects in their diets 14. Consequently, UGT-catalyzed biotransformation of xenobiotics has been implicated in some cases of insecticide resistance 17. In addition, insect UGTs play important roles in several processes, including cuticle formation, pigmentation, and olfaction 181920. However, only limited molecular information on insect UGTs is available.

Genome sequencing projects offer a new route into understanding multigene families both within a single species and across different species. In the present study, we have used the newly assembled 9× coverage genome sequence http://silkworm.swu.edu.cn/silkdb/ to characterize the UGT multigene family in silkworm. In total, the 42 putative silkworm UGTs were identified. The phylogenetic relationships among these genes and the homologs from the sequenced insect genomes were analyzed. Searching available ESTs and microarray data, we found that 36 of 42 silkworm UGTs were expressed in different tissues, suggesting that these genes are active and may have different functions. In addition, the genomic structures of silkworm UGT genes were also investigated. Our data provide the preliminary insights into evolution and functions of the silkworm UGTs. This is the first time to describe this gene family in a Lepidoptera species. The results may have important implications for the study of insect UGTs.

Taking advantage of the newly assembled silkworm genome sequence, we identified the 42 putative members of UGT genes, including a reported silkworm UGT gene. The number of UGT genes in silkworm is larger than D. melanogaster UGTs, which contain 33 genes 21. While in plant of Arabidopsis there are about 120 UGTs and mammalian UGT gene superfamily currently has 117 members, they are all quite larger number than insects 22.

Both plant and mammalian UGT sequences contained a signature sequence–a UDP-glycosyltransferase signature: (FVA) - (LIVMF) - (TS) - (HQ) - (SGAC) - G - X(2)- (STG) -X(2)- (DE) - X(6) - P - (LIVMFA) - (LIVMFA) - X(2) - P - (LMVFIQ) -X(2)- (DE) - Q (all amino acids that can occur at a given position are listed inside brackets; X indicated any amino acid 11). It was reported that this motif has been identified in a range of prokaryotic and eukaryotic organisms. There is no exception of insects UGTs. The signature motif is located on C-terminal of the protein sequence.

So far all the identified UGTs comprise two major functional domains 23. The N-terminal half, believed to be responsible for binding the aglycone, tends to be less conserved than the C-terminal half, which is thought to bind the UDP-sugar. Aglycones bound by UGTs are highly diverse, hence the low amino acid sequence conservation in the N-terminal region between the members of this family. The silkworm UGTs followed the same pattern, with the C-terminal half showing the highest similarity to other UGTs.

The best-characterised UGTs are the mammalian UDP-glucuronosyltransferases, which transfer glucuronic acid to hydrophobic substrates. These enzymes localized in the lumen of the endoplasmic reticulum and are membrane-bound 24. Mammalian UGTs have two functional motifs are thought to be important for the topology of proteins within the cell. One is the mammalian UGTs contain the N-terminal signal sequence cleaved on cotranslational segregation into the endoplasmic reticulum 2526. The other is the putative hydrophobic transmembrane domain located near the carboxyl terminus of the protein, this domain anchors the enzymes to the membrane region 27252829. The major portion of the protein is located in the ER lumen, including the proposed substrate-binding domains and the catalytic site. The silkworm UGTs also have these domains, an N-terminus signal sequence and hydrophobic transmembrane domain either in N-terminal or C-terminal or in both terminals. Thus, these genes are most likely to be anchored in the endoplasmic reticulum. However, no such motif was identified in Arabidopsis UGTs 22, supporting that the plant UGTs are cytoplasmic enzymes, different from insect and mammalian UGTs.

42 members of the silkworm UGT gene family scattered on 10 chromosomes. Phylogenetic analysis of these genes defined 5 consistent groups. The genes on each cluster often show a high degree of sequence similarity. This suggests that several gene duplication events took place during the evolution of this family. These duplication events included both tandem events, where the duplicated copies remain adjacent to each other, and transpositional events, where one copy is translocated to a different chromosomal location. Examples of both types of event can be seen in the clade including BmUGT007327, BmUGT002854 and BmUGT009788.

At least 180 introns were identified in 42 silkworm UGTs; the average introns number of each gene is 4.5. However, in D. melanogaster each UGT genes have 1.8 introns, quite smaller number than silkworm UGTs; the size of UGTs from the two organisms are similar, all about 500 amino acids. The increase of number of introns in silkworm UGTs is probably due to a fact that the silkworm genome harbors a large proportion of repetitive sequences. And the majority of these repetitive sequences were transposable elements or the remainders of transposable elements. Insertions of transposition into the introns might result in the increase of their lengths.

The micoarray data and RT-PCR results from fifth-instar day 3 larvae tissues showed that the silkworm UGT genes exhibited widely different patterns of expression. Different expression profiles indicate that these UGT genes might have different functions. BmUGT001338 and BmUGT003817 were expressed in all most tissues and different developmental stages according to microarray data, this indicated that these genes might have important functions in silkworm development and play the housekeeping role. The RT-PCR results showed that BmUGT010286 were transcripted in all the tissues, just a low expression was observed in head, silk glands, testis and ovary and possessed similar tissue expression pattern with BmUGT1, which reported play a major role in detoxication responses 30. In addition the two genes located on the same chromosome and may have a common ancestor and phylogenetic tree showed that they were classified into a sub-group. These indicated that BmUGT010286 might involved in the detoxication of plant allelochemicals. B. mori larvae take up flavonoids into their cocoons from the leaves of their host plant, the mulberry tree (Morus alba) 31, but the flavonoids in mulberry tree leaves were different from that which isolated from the cocoon shell of the silkworm 32. In insects, the formation of glucoside is the predominant pathway for dietary flavonoids 33343536, and the glucosylation of polyphenolics in insects is catalyzed by UDP-glucosyltransferase (UGT) 1415, B. mori UGTs changed the flavonoids glucose conjugation positions from 3-O-glucoside to 5-O-glucoside or other forms to increase fitness for their own. Since a flavonoid 5-O-glucoside has not yet been reported in plants to other animals. Transfer a glucose moiety to the C-5 position of the flavonols, is functioning in B. mori 37. It also was reported that quercetin 5-O-glucoside was the predominant metabolite in the midgut tissue, while quercetin 5,4'-di-O-glucoside was the major constituent in the haemocyte and silk glands 37. The genes were highly or specific expressed in silk glands, midgut and haemlymph such as BmUGT010289, BmUGT004965, BmUGT013859, BmUGT003835, BmUGT013829, BmUGT013830, BmUGT013860, BmUGT013834, BmUGT010286 might have functions in flavonoids metabolism in silkworm. This may be a very important function for silkworm and further study should be needed to confirm this inference in future. The RT-PCR results also indicted that BmUGT003835 gene was highly expressed in testis, ovary, integument, fat body, midgut, malpighian tubules and with lower expression in head, but hardly detectable in haemocyte and silk glands. This expression profile suggested this gene might have some functions in detoxication since the intergument, fat body, and midgut are the main tissues correspond to such activity. We know little about what roles this gene plays in the testis and ovary. It is interesting and worthy of further study. In D. melanogaster, there were several UGT genes involved in olfaction; they were preferentially expressed in the third antennal segment of D. melanogaster 20. Both microarray data and RT-PCR indicated that BmUGT013829 has high expression levels in silkworm head and RT-PCR also shows that this gene is highly expressed in larval and adult antennae (Figure 7B), suggesting that this gene may be involved in olfaction, but the expression of this gene is not antennae-specific. It can be also detected in fat body and integument, suggesting this gene may have other functions. With more and more insects UGT genes were identified, individual silkworm UGTs functions can initially be determined through bioinformatic studies that reveal homology to genes encoding enzymes of known catalytic activity.

It is known that many plant phenolics can act as toxins or feeding deterrents to insects and thus play an important role in plant defense against herbivorous insects. The detoxication of ingested plant phenolics is believed to be one of the principle functions of insect UGT enzymes 14. Compared with A. gambiae, D. melanogaster, and A. mellifera UGTs, B. mori has more UGTs. Probably this is the result of competition between silkworm and its only diet mulberry leaf. In order to defense B. mori, mulberry leaf can produce some toxicant chemicals, while B. mori can also evolve some mechanism to detoxicate the chemicals. UGTs probably are involved in this process, so the number of the silkworm UGTs was expanded and also produce the silkworm-specific UGT genes.

Biochemical evidence and comparisons with mammalian and other systems point to a range of important functions for the UGT genes of this family. Our results indicate that the B. mori contains the largest insect UGT gene family characterized to date compared with other insects. The data presented in this study provide an overview for the silkworm UGTs and facilitate their functional studies in future.

FFH carried out the analysis of the sequences and drafted the manuscript. CLC revised the manuscript. ZZ improved the study design and revised the manuscript. ZHL helped to perform the experiment of RT-PCR. FYD revised the manuscript. CL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. ZHX supervised the study. All authors read and approved the final manuscript.