Study On The UDP-glucosyltransferase Superfamily In The Silkworm, Bombyx Mori

Glycosylation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds.A class of UDP-glycosyltransferases(UGTs,EC 2.4.1.₎ is involved in this process.UGTs are a superfamily of multifunctional enzymes found in almost all living organisms,including animals,plants,bacteria and viruses.UGTs 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-glucose,UDP-galactose,UDP-xylose and UDP-glucuronic acid.UGTs mainly classied into severa kinds,including plant UGTs, mammalian UGTs and insect UGTs.Plant UGTs using UDP-glucose as glycosyl donor.They make glucosidation on a number of chemicals including plant hormones,secondary metabolites and xenobiotics such as pesticides.The mammalian UGTs using UDP-glucuronic acid as glycosyl donor, they play important role in the detoxification of xenobiotics.In addition,they involved in regulating the metabolism and balance of many endogenous substrates,including bilirubin and steroids.Like plant UGTs,insect UGT enzymes also use UDP-glucose rather than UDP-glucuronic acid as sugar donor.Also,similar to the mammallian,both endogenous and exogenous substrates are subject to glucosidation in insects.Insect UGTs play an important role in detoxication of plant allelochemicals encountered by many insects in their diets.In addition,insect UGTs play important roles in other several processes,including cuticle formation,pigmentation,and olfaction.Ecdysteroid UDP-glucosyltransferase that is egt gene is a special glycosyltransferase gene which is encoded by insect baculovirus.The EGT enzyme produced by baculovirus after infecting insects can transfer the glycosyl from UDP-glucose in insect to ecdysteroids,resulting the inactivity of ecdysteroids,further blocking the moulting and metamorphosis of insect host,so the virus can have longer time to yeild more progenies.Lepidoptera insect is the main pest in agricultural field.Bombyx mori is the model organism of Lepidoptera and also is the only organism of genome sequencing in Lepidoptera.Thus,study on silkworm UGTs at genomic level is not only provided molecular foundation for the function study of silkworm itself,but also established important reference for studing other insects UGT genes.Our study use bioinformatics methods to analyze the Bombyx mori UGTs with the silkworm genome sequences,ESTs and microarray data.Meanwhile we perform further function study on one of these UGTs genes BmUGT013829 by the technology methods of gene clone,RT-PCR, prokaryotic expression,immunohistochemistry,and eukaryotic expression and so on.The main aviable results are as follows:1.The bioinformatics analysis of Bombyx mori UDP-glucosyltransferase and the research of their expression pattern.Using the assembled 9x coverage genome sequence to characterize the UGT multigene family in silkworm by bioinformatics analysis.In total,42 putative silkworm UGTs were identified.Based on the EST and microarray data,there are 36 UGTs genes have expression evidence.Among 42 silkworm UGTs,two genes were reported previously,the other 40 genes were first identified by our research.Compared with other insects,the silkworm UGTs number were greatly expanded(using the same method we identified 22 UGTs from Anopheles gambiae genome,12 UGTs from Apis mellifera genome,and there are 33 UGTs in Drosophila melanogaster genome),Phylogenetic analysis shows that silkworm has its-specific UGT classes,there are Groupâ… ,Groupâ…¡andGroupâ…¤. The reason why the number of the silkworm UGTs was expanded and also produce the silkworm-specific UGTs genes is probably the result of long competition between silkworm and its only diet mulberry leaf.All the identified silkworm UGTs comprise two major functional domains,The N-terminal half and the C-terminal region.The N-terminal half tends to be less conserved than the C-terminal half, which is the same with the earlier research on the other species UGTs.42 members of the silkworm UGTs genes family scattered on 10 chromosomes.Phylogenetic analysis of these genes defined 5 consistent groups,there are groupâ… -â…¤.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.Relative to Dipteran UGTs such as D.melanogaster,silkworm UGTs contained more and larger introns.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.Analysis of the ESTs data indicated that among all the UGT genes there are 24 genes having ESTs evidences.These genes have transcriptive activity were mainly in midgut,haemocyte,testes, ovary,silk glands tissues and some genes have EST evidence in diapause eggs.Based on the microarray data,29 UGTs genes have probe sequence.We construct the cluster picture of the different tissues microarray data on these 29 UGT genes.And most genes exhibited widely different patterns of expression,16 genes have transcriptive activity in midgut. BmUGT003817 and BmUGT003835 genes express widely in silkworm tissues.Some genes were expressed in the tissue-specific pattern,such as BmUGT004965 and BmUGT010289 were only expressed in silk glands.The RT-PCR results were similar to the microarray data detected.BmUGT013835, BmUGT013859,BmUGT013830 and BmUGT010286 genes were widely expressed in 3-day-old fifth-instar larvae tissues.BmUGT013860 and BmUGT013834 genes were only expressed in midgut tissue,and BmUGT004965 and BmUGT010289 were only expressed in silk glands tissue.Both the microarry data and the RT-PCR results show that the silkworm UGTs genes show different expression patterns.The UGT genes of different classes have different tissues distribution. While the genes on the same group the expression pattern are slso different,even though the gene duplication event taken place.The different expression profiles indicate that these UGTs genes might have different functions,2.Cloning,sequence analysis and expression pattern of silkworm BmUGT013829 geneWe cloned a UGT gene named BmUGT013829 which located on the 28 chromosome,and then gained the complete CDS.Making sequence analysis and the relative predication on the cloned gene indicated that this gene has four extrons,the length of the BmUGT013829 CDS is 1545 bp and it encodes 514 amino acids,with the predicted molecular weight of 57.5 kDa and isoelectric point of 6.41.Further analysis show that this gene has a signal sequence composed of 16 amino acids from 1 to 16,and a putative hydrophobic transmembrane domain from 477 to 499 amino acids residue.An alignment of this gene protein sequence on the NCBI revealed it has the highest homology with the silkworm UGTs gene(Accession No.AF324465),sharing 76%similarity in amino acid level,and then shares 61%similarity with the tribilum antennal-enriched UGTs gene.Microarray data show that BmUGT013829 gene has expression in the head,fat body and integument tissues of silkworm larval,and with the high level in head tissue.The RT-PCR result show that BmUGT013829 gene has the high expression level in the head,fat body and integument tissues,and also show this gene has very high expression level in laravl and adult antennae,which indicates that this gene is an antennal-enr(?)ched UGTs gene,may involved in olfaction.In addition, this gene was also deceted miner expressin in midgut,silk glands,ovary and testis tissues.Detection on transcriptional level indicate that BmUGT013829 gene expression pattern is not antenna-specific, These indicate that this gene might have other functions besides involving in olfaction.3.Prokaryotic expression and preparation polyclonai antibody of silkworm BmUGT013829The complete ORF of BmUGT013829 was subcloned into the pET-28a-c(+) expression vector,the recombinant plasmid was transformed into Escherichia coli BL21 and then induced BmUGT013829/PET-28a/BL-21 to express protein by IPTG.We got a recombinant protein with molecular weight about 60kD,The target protein molecular weight is 57.5kD.Addingthe six-His tag protein so the totle molecular weight is up to about 60kD,which is consistent with the prediction. The protein is expressed as inclusion bodies by appraisal.Purify BmUGT013829 protein by the method of affinity chromatography and electro eluter together,then inject rabbit,and prepare polyclonal antibody.Western blotting analysis of the BmUGT013829 protin expression pattern in 3-day-old fifth-instar larvae tissues.The result indicate that BmUGT013829 protein have expression in fat body,head,integument and midgut,only with very lower expression level in midgut.Experiment result show that BmUGT013829 have expression both on mRNA level and protein level.,and both the levels are consistent.Furthermore Western blotting result also shows the antibody is ideal,which can detect BmUGT013829 protein.4.Immunohistochemistry of silkworm BmUGT013829 proteinUsing established parafin section technical method,we make parafin section on fifih-instar day 3 larvae head tissue.After did the H.E coloration,we can see the components of the head clearly,this can provide the foundation for analysising the following experiment result.Immunohistochemistry result indicating that BmUGT013829 protein expressed in all components of head tissue,such as head muscle,but it has higher expression level in antennae than in other constituents.Thus BmUGT013829 is a antennal-enriched UGT gene may involved in olfaction.Otherwise it might has other functions,such as in detoxication responses,because BmUGT013829 protein have high expression level in integument and fat body(Western blooting result),since they are the main detoxication areas for silkworm.5.Eukaryotic expression of silkworm BmUGT013829 gene and activity determinationWe subcloned the BmUGT013829 gene into the pFastBac^TMHT-B eukaryotic expression vector and then recombined into Bacmid gained the recombinant plasmid of Bacmid-BmUGT013829,which is use to express the recombinant protein of BmUGT013829 in insect cells.In order to determin if BmUGT013829 protein do express,we did the western blotting to verify.The results indicate that BmUGT013829 protein was successfully expressed in sf9 cells. We used P3 generation of recombinant baculovirus to infect High Five cells.By using wild Bacmid as a control.After 48h High Five cell pellet were used for activity determination.We detected the BmUGT013829 activities toward severl substrates belong to different chemical groups. The thin layer chromatography results show that BmUGT013829 can catalyze the glucosylation of some odorants,such as vanillin and guaiacol,indicating BmUGT013829 is involved in olfaction. In addition,BmUGT013829 can also catalyze the glucosylation of phenolics and phenol-derived compounds,including flavonoids such as 1-Naphthol,quercetin 3-β-D-glucoside,suggesting BmUGT013829 also has the role in detoxication response.