Cloning,Expression And Function Of Male Antennae-specific BmGSTD4in The Silkworm, Bombyx Mori

Glutathione-S-transferases(GSTs, EC2.5.1.18)are a family of multifunctional enzymes,which were found in mammals,insects,plants, bacteria and fungi.GSTs play a key role in the detoxification of both endogenous and xenobiotic compounds. Besides,GSTs are also involved in protection against oxidative stress, isomerase activities and modulation of signaling pathways. Some GSTs also have ligandin (transport) functions. A class of olfactory-related GSTs were identificated in Cattle, rats, Manduca sexta, Helicoverpa armigera and Papilio xuthus,were supposed play a role in the olfactory systerm.Bombyx miro Delta GSTs shared more than70%identity with Manduca sexta GST-molfl,which is olfactory-specific,the two genes are clustered into a cluster of evolutionary analysis. BmGSTd4did’t have full-length CDS sequence,and less reported. Based on the above clues,so we guess if BmGSTd4have the functions related to the olfaction?The main results are as follows:1. Expression pattern of the BmGSTd4geneThe expression paterms of BmGSTd4gene in different tissues and developmental stages were analysised.The results showed that BmGSTd4gene wasn’t expressed in various tissues of day3of5th instar larva.but expressed exclusively in the male antennae suggesting it may be involved in the olfaction.2. The cloning of the full-length cDNA of BmGSTd4gene and sequence analysisBombyx miro Delta GSTs have four members.downlaoded the uncompleted sequence of BmGSTd4gene from NCBI GeneBank. made a prediction of domain. cDNA reverse transcripted from male moth antennae total RNA as templates in Dazao strain of Bombyx miro,we used RACE to obtain225bp5’fragment and221bp3’fragment,through compared the two frangments with sequence predicted from Yu, eliminated overlapping sequences, spliced and obtained BmGSTdA cDNA sequence with a full-length of1049bp containing an entire open reading frame(ORF). The sequence are composed of738bp coding region,90bp5’UTR and221bp3’UTR. The sequence encods497amino acids, the molecular weight of the protein is27kDa, of which the isoelectric point is4.66.The BmGSld4gene is located in nscaf2853on chromosome6of silkworm,include one intron and one extron.The protein possess Integrity domains. The first22amino acids are signal peptide.Compared BmGSTd4and other species of GSTs downloaded from NCBI have been reported using.Clastalx,then constructed phylogenetic tree using the MEGA5.The results showed that BmGSTD2and BmGSTD3clustered together, the genetic evolutionary relationship similar to the olfactory gene from Papilio xuthus. BmGSTD1and BmGSTD4clustered together, similar to the olfactory specific gene from Manduca sexta and Helicoverpa armigera.3. Prokaryotic expression and preparated antibody of BmGSTD4Because of having signal peptide,and in order to improve the prokaryotic expression, so truncated N-terminal24amino acid residues of BmGSTD4for expression. We successfully constructed BmGSTd4-p28recombinant vector with the N-terminal His tag, inducible expression in Rosetta (DE3) expression strain successfully.Through Ni-NTA purified the protein BmGSTD4,then desaltied and concentrated, get500μL13μg/μL recombinant protein.Take200μL BmGSTD4protein preparating for the commercialization of polyclonal antibody, antibody titer is1:100000.4. Localization and functionWestern blot validated the specificity of BmGSTD4in protein level, demonstrated that BmGSTD4is expressed in the antennae of male moth specially.Immunofluorescence localization of BmGSTD4in antennae of male moth showed that BmGSTD4protein is localized in the hair sensor.Using1-chlorine-2,4-nitrophenyl(CDNB) as a substrate.we measured the absorbance diversify in340nm.Then we got the standard activity curve through sofeware.The kinetic parameters Km and Vmare0.020±0.001(mM).176.302=4.943 (μmol/min/mg), demonstrated BmGSTD4protein had good activity.PT-PCR,western blt demonstrated the antennae-specificity for BmGSTD4, Immuno fluorescence localization of BmGSTD4showed it was existed in hair sensor. BmGSTD4protein had good activity.All results suggested BmGSTD4play a role of protecting the olfactory systerm from harmful xenobiotics.And another functions about alfaction need next verify.