Isolation And Expression Analysis Of Midgut Protein Cdnas From Holotrichia Parallela Motschulsky Larvae

Holotrichia parallela Motschulsky which belongs to the family Motschulsky, is oneof the most destructive agricultural and landscape pests in the world, because of theirability to damage peanuts, beans and food crops. Peanut is an important oil and cash cropworldwide, however, severe reductions (10%~30%) in peanut yields are caused by whitegrubs, the subterranean larvae of scarab beetles. Grubs are now recognized as the mostimportant pests of peanut worldwide because of their ability to damage roots.Furthermore, Grub control in China is difficult due to the underground livingenvironment. Therefore, it is necessary to develop a more effective and environmentalfriendly pest control method. Peritrophic membrane (PM) is a physical barrier, protectingthe midgut epithelium from abrasive food particles, digestive enzymes and pathogensinfectious peros. PM became to target substrate and the understanding of the interactionbetween peritrophic membrane and pathogenic microorganism is more important forbiological control. In this study,350of clones were abtained by screening the cDNAexpression library of H. parallela larvae midgut,3full-length cDNA genes of them werecharacterized based on the clone sequencing and expression analysis. Summarization isshown as follows:The quality and quantity of RNA and mRNA was purified from H. parallela midgut,and midgut cDNA expression library of H. parallela was constructed successfully, thislibrary has a capacity of1.8×108pfu/mL, its recombination efficiency was98.8%, andthe insert sizes were about1.8kb based on PCR analysis.350positive clones wereobtained by immunoscreening the cDNA library with an anti-PM proteins polyclonalantiserum, three cDNA clone were obtained by bioinformatics analysis: hpcbp45geneencodes chitin binding protein HpCBP45; hpcda5encodes chitin deacetylase HpCDA5,and hpce19gene encodes carboxylesterase HpCE19.hpcbp45gene encoded the chitin binding protein. The full length of open readingframe of hpcbp45is1923bp, and encodes641amino acids. It was predicted that therewas a signal peptide in consist of21amino acids at the N terminal of the protein with apredicted molecular weight of69.5kDa. Analysis by prosite database and blast byDNAMAN revealed that it consisted of8tandem putative chitin binding domains with conserved sequence motifs CX13-22CX5CX9CX12CX7C and belong to the peritrophin-Adomains. The hpcbp45were successfully expressed69kDa protein after IPTG induction.Western blot analysis showed that the yeast recombinant protein with an apparentmolecular weight of100kDa, significantly higher than the predicted molecular weight of69.6kDa. The recombinant protein HpCBP45belong to class3PM protein exhibited itsactivity to bind chitin, The chitin-bound protein could only be released from the chitin by1%Calcofluor and6M urea. The relative expression expression of hpcbp45gene wasexamined by qRT-PCR, HpCBP45was primarily synthesized in the midgut.hpce19gene encoded the carboxylesterase, The full–length ORF of HpCE19(1446bp) encodes534amino acids with the predicted molecular weight of59.2kDa andpI of4.19. Prosite and PROSCAN programs scanning of the protein sequence revealedthe highly conserved Esterase/lipase superfamily sites from amino acid residudes23to507and the carboxylesterases type–B serine active site FGGnpdsVtIsGqSAG. Proteinactive sites includes three amino acid residues a catalytic triad consisting of a serine, anaspartate and a histidine residue (Catalytic triad: S203, D334, H438). The protein hasthree predicted N–glycosylation sites based on ExPASy Proteomics Server located atamino acid residues257、408and448. Western blot analysis showed that HpCE19wassuccessfully expressed in Pichia pastoris GS115. Carboxylesterase activity assay in vitroshowed that the recombinant HpCE19could hydrolyze α–naphthyl acetate (α–NA).Analysis of tissue–dependent expression revealed that the transcript is primarlyexpressed in the midgut by qRT–PCR approach, which provides a foundation for thefurther research of function of H. parallela carboxylesterase HpCE19.hpcda5gene encoded chitin deacetylase, The cDNA hpcbp45(GeneBank AccessionNo. KC703972) containing an ORF of1137bp. The deduced protein sequence showedthat HpCDA5was synthesized as a preprotein of379amino acid residues with thepredicted molecular weight of42.2kDa, pI of4.08and a19-amino acid signal peptidepredicted by the software SignaIP. The protein has a polysaccharide deacetylase-likedomain and three predicted N–glycosylation sites based on ExPASy Proteomics Server.Search of the GenBank database using blastp programs showed that HpCDA5hadsequence similarity with TcCDA. The hpcda5was successfully expressed in insect cells(sf9) as secreted proteins using recombinant baculoviruses (Bac to Bac system). Westernblot analysis showed that the apparent molecular weight for recombinant HpCDA5wereabout42kDa. Subsequent chitin binding assays demonstrated that HpCDA5had chitinbinding afnity. qRT–PCR approach revealed that hpcda5has high expression in eggs,but not detected in the head. We also detected that the expression of hpcda5gene washigher after fed the larve with cry8Ea3toxin, and return to normal at8hours, suggested that the upregulation of hpcda5gene after fed with Cry protein, hypothesize that thisprotein might be responsible for the homeostasis of the PM structure.