Isolation And Identification Of Midgut Target Proteins From Helicoverpa Armigera (Hübner)

The Helicoverpa armigera (Hübner) is an important Lepidopteran pest in the world. It can damage more than 200 different sorts of plants including cotton, wheat, maize, broomcorn, tomato, beans, melons, and so on. The peritrophic matrix (PM) is a physical barrier protecting the midgut. The recent PM model predicts that may be targeted by various mechanisms. Further understanding of the molecular structure and function of PMs will provide new opportunities for development of midgut targeting strategies for biocontrol of insect pests. In this study, the H. armigera larval midgut cDNA expression library was immunoscreened with an anti-PM proteins polyclonal antiserum to abtain 385 positive clones. 26 purified cDNA clones of them were chosen for sequencing then three full-length clones, hm72, hc3 and ha8 were sequenced.The hm72 gene encoded H.armigera IIM (GenBank accession number is HM017910) was full-length clone with 2 888 bp, and the longest open reading frame was 2 448 bp. HM72 preprotein had 816 amino acids with N-terminal contained a signal peptide which was composed of 22 amino acids. The HM72 with isoelectric point of 3.69 is rich in Gly(G), Asp(D) and Glu(E). The structure analysis showed that HM72 contained 5 chitin binding domains (CBDs), 1 mucin domain and 2 D-G rich regions. By Western blotting analysis, we not only found the protein (HM72) PM and midgut tissue but the protein was found in exuviae and fecal pellets, especially rich in the entire of midgut. However, we did not find it in other tissues and structures. Based on the 760 to 1281 nucleotide of hm72 gene sequence, a pair of primers was designed to synthesized double-strand RNA (dsRNA) to silencing the hm72 gene but there was no significant effect.The hc3 (GenBank accession number is GU119888) encoded H.armigera carboxylesterase. that full length was 2 896 bp, and encoded 795 amino acids. Isoelectric point of HC3 was predicted to be 3.94. The HC3 possesses a catalytic triad of Ser204, Glu331 and His444. The amino acids (545 to 773) of HC3 sequence contained highly repeated GDE domain which constituted a hydrophilicity profile. An engineering strain E.coli BL21/HC3 was constructed by transformation of a recombinant plasmid pET21b-hc3. The HC3 was successfully expressed in the engineering strain by induction of 0.6 mM IPTG. The gene (GenBank accession number is HM012573), ha8, was cloned and analyzed, which was a full-length of ha8 was 2 353 bp, and encoded 832 amino acids containing of a signal peptide consisting of three amino acids only because of incomplete N-terminal and there was no initiation codon. HA8 was proved to be an Argonaute protein which contained an intact PAZ domain profile and Piwi domain profile. The successful prokaryotic expression was performed when ha8 was recombined into pET21b/pET28b vector.