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Molecular Cloning, Expression And Characterization Of Hatching Enzyme Gene In Lepidopteran Pests, The Rondotia Menciana Moore And Plutella Xylostella

Posted on:2015-02-06Degree:MasterType:Thesis
Country:ChinaCandidate:H Y WangFull Text:PDF
GTID:2283330422488628Subject:Biochemistry and Molecular Biology
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Rondotia menciana Moore and Plutella xylostella are lepidopteran pests. Rondotiamenciana Moore, one of main pests in the mulberry fields, is belonged to a genus of rondotiaof the bombycidae family, Lepidoptera. And Plutella xylostella is belonged to plutellidaefamily, Lepidoptera. It is a worldwide major agricultural pests. In this paper, we used thesetwo pests as research materials, obtained the full-length cDNA sequence of RmHE and PxHEby the method of RACE-PCR and SMART cDNA synthesis technique.The full-length cDNA sequence of RmHE is1325bp in length, and contains an integralORF of885bp, encoding294amino acids residues which includes a putative signal peptideof16amino acids and a mature protease of208amino acids.The molecular weight of RmHEis33.41kD and it’s isoelectric point is5.80. The amino acid sequence from92to242ofRmHE is the zinc metalloproteinase domain. Homologous analysis results of the deducedprotease domain showed that RmHE has83.8%–30.1identities to that of HE identified in theother species. And RmHE has the highest homology with BmandHE compared with otherspecies. The full-length cDNA sequence of PxHE is938bp in length, and contains anintegral ORF of882bp, encoding293amino acids residues which includes a putative signalpeptide of16amino acids and a mature protease of208amino acids. The molecular weight ofPxHE is33.23kD and it’s isoelectric point is4.99. The amino acid sequence from91to241of PxHE is the zinc metalloproteinase domain. PxHE has79.2%–29.1identities to that of HEidentified in the other species. And PxHE has the highest homology with PcrHE comparedwith other species. Bioinformatics analysis indicated that two similar signature sequences ofHE, HExxHILGFLHMQSTYNR and YDYVSCLHY, were harbored in RmHE and PxHE.The SWISS-MODEL software predicted that the three-dimensional structure of RmHE andPxHE both are similar to that of zebrafish hatching enzyme.Changes of RmHE transcripts occurred in accordance with the process of embryonicdevelopment, and reached to the maximum at the hatching time, which indicated that it mayplay an important role in the process of embryonic development and hatching. Moreover,RmHE transcripts could be detected in the midgut at larval stage, and the western blot resultof detecting each tissue protein indicated that RmHE specificly expressed in the midgut as themature form, which suggested that RmHE may have the biological function of food digestion. PxHE gene has only one exon, unlike the multi-exon gene structure for BmHE. the corepromoter of PxHE gene was predicted, which located in the-52to-3bp position fromtranscription start site in5’-upstream region. This region contained not only basal promoterelements, such as TATA-box and GC-box, but also some transcription factor binding sites,closely related with embryo development, such as HSF1and GATA-1. Homology analysisshowed that four cysteine residues were conserved in all Astacin Family proteases. Thephylogenetic tree showed that bombycidae family, saturniidae family and plutellidae familyare obviously clustered in different branches.Furthermore, we cloned the corresponding gene sequence of ORF, removing signalpeptide, mature enzyme of RmHE and PxHE into prokaryotic expression systems for proteinexpression. Western Blot results verified that the proteins of RmHE and PxHE highlyexpressed. The results of this study provide a molecular basis for further clarifying thehatching mechanism of Rondotia menciana Moore and Plutella xylostella, and provide basicinformation for developing new insecticides which the hatching enzyme as a target.
Keywords/Search Tags:Rondotia menciana Moore, Plutella xylostella, Hatching enzyme gene, Molecularcloning, prokaryotic expression
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