Construction Of CDNA Expression Library Of Holotrichia Oblita Larvae And Identification Of Its Peritrophic Membrane Target Proteins

Holotrichia oblita is one of the most destructive agricultural and landscape pests in the world, and belongs to the family Scarabaeidae (Coleoptera, Scarabaeidae). Peritrophic membrane is a physical barrier, protecting the midgut epithelium from abrasive food particles, digestive enzymes, and pathogens infectious peros. Identifying and characterizing of the peritrophic membrane proteins can lay a solid foundation for its using as biological control targets and the understanding of the interaction between peritrophic membrane and pathogenic microorganism. We have constucted a cDNA expression library of the Holotrichia oblita midgut, identified and chacterized several peritrophic membrane target proteins. Main achievements summarized as follows:1. Midgut RNA and mRNA were isolated from the Holotrichia oblita larvae using the RNeasy total RNA isolation kit and the Oligotex mRNA isolation kit, and a cDNA library was constructed from Holotrichia oblita midgut mRNA using the ZAP-cDNA Gigapack Cloning Kit.The cDNA was unidirectionally ligated into the Uni-ZAP XR vector. The library had a complexity of 5.18×10~6 plaques, of which over 98.8% were recombinants. Titer of the amplified library was 2.36×109pfu/mL, and the average length of the inserted fragments was about 1.85kb by PCR analysis. About 254 positive clones were obtained by screening the library with an aitiserum specific to the PM protein of Helicoverpa armigera, including HoCBP2, HoCBP76, HoSCP, HoChi and HoIIM.2. HoCBP2 and HoCBP 76 encoded the chitin binding protein. The cDNA HoCBP2 (GeneBank Accession No. JF681185) was 2226 bp in length, containing an ORF of 1947 bp. The deduced protein sequence showed that HoCBP2 was synthesized as a preprotein of 649 amino acid residues with the predicted molecular weight of 70.2 kDa, pI of 3.52 and a 19-amino acid signal peptide predicted by the software SignaIP. The cDNA HoCBP76 (GeneBank Accession No. GU12810~6) was 2019 bp in length, containing an ORF of 1725 bp, The deduced protein sequence showed that HoCBP76 was synthesized as a preprotein of 575 amino acid residues with the predicted molecular weight of 62.3 kDa, pI of 3.51 and a 19-amino acid signal peptidel. Search of the GenBank database using blastp programs showed that HoCBP2 and HoCBP76 had sequence similarity with PMP14 (GeneBank Accession No. GU12810~6) from Tribolium castaneum (Coleoptera: Tenebrionidae), the identity were 35% and 34% respectively. Deduced amino acid sequence analysis indicated that HoCBP2 and HoCBP76 contained eight and seven tandem putative chitin binding domains which belonged to the peritrophin-A domains, respectively. The HoCBP2 and HoCBP76 were recombined into pET21b vector, transformed into E.coli., and were successfully expressed after IPTG induction.Recombinant HoCBP2 and HoCBP76 were successfully expressed in insect cells (Tn-5B1-4) as secreted proteins using recombinant baculoviruses. Western blot analysis showed that the apparent molecular weight for recombinant HoCBP2 and HoCBP76 were about 120 and 110kDa. Subsequent chitin binding assays demonstrated that both of the recombinant HoCBP2 and HoCBP76 had chitin binding a?nity. The HoCBP2 and HoCBP76 tightly bound to chitin and did not dissociate from the chitin following treatment with PBS and 1 M NaCl. However, they were solubilized from the bound chitin by 6M Urea or by 1% Calco?uor. Immunolocation analysis using the antibodies reacting to HoCBP2 and HoCBP76 showed that the abundance of HoCBP2 and HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, HoCBP2 and HoCBP76 was mainly present in the peritrophic membrane, midgut and ovum, some weak postitive staining was detected in the extract from the fecal pellet, integument, digesive fluid, fat body, exuviae and Malpighian tubules extracts did not show any positive reaction.3. HoChi encoded the chitinase protein. The cDNA was 1636bp in length (GenBank Accession No. HM596340), containing an ORF of 1458bp, and flanked by a 5'untranslated region of 27bp and a 3'untranslated region of 145bp. The deduced protein sequence showed that HoChi was synthesized as a preprotein of 486 amino acid residues with the predicted molecular weight of 51.8 kDa, pI of 5.58. A potential polyadenylation signal sequence AAGAAA was located at 39bp upstream of the polyA tail. It shared the typical character of chitinase protein, including a signal peptide, a chitinase active site, a C-terminal threonine-rich region and a chitin-binding domain, belonging to the chitinases family 18. The deduced amino acid sequence showed a high identity to reported chitinases from other insect, especially the Coleopteran insect Tribolium castaneum (GenBank Accession No. NM001044629). The HoChi was recombined into pET28b vector, and transformed into E.coli. Western blot analysis demonstrated that HoChi protein was successfully expressed after IPTG induction, and recombinant HoChi was successfully expressed in insect cells (Tn-5B1-4) using recombinant baculoviruses.4. HoSCP encoded the serine carboxypeptidases protein. The cDNA was 1830 bp in length (GenBank Accession No. JF681184), containing an ORF of 1830 bp, and flanked by a 5'untranslated region of 78bp and a 3'untranslated region of 381bp. The deduced protein sequence showed that HoSCP was synthesized as a preprotein of 457 amino acid residues with the predicted molecular weight of 51.2 kDa, pI of 6.12 and a 15-amino acid signal peptide. A potential polyadenylation signal sequence AATAAA was located at 14bp upstream of the polyA tail. Deduced amino acid sequence analysis showed that HoSCP contained an eonserved peptidase S10 domain, the serine active site IAGESYAG, the histidine active site AFLkVYGAGHMVPMDQP, and two potential sites for N-glycosylation. The sequence also indicated the presence of a catalytic triad (Ser-185,Asp-361,His-418). The HoSCP was recombined into pET28b vector, and transformed into E.coli. Western blot analysis demonstrated that HoSCP protein was successfully expressed after IPTG induction, and recombinant HoSCP was successfully expressed in insect cells (Tn-5B1-4) using recombinant baculoviruses.5. HoIIM encoded the insect intestianl mucin protein. The cDNA was 1879 bp in length (GenBank Accession No. JF JF681187), Deduced amino acid sequence analysis indicated that the HoIIM protein contained five tandem putative chitin binding domains which belonged to the peritrophin-A domains, and five mucin domains which riched in threonine, proline and serine, and also contained several repeating units, PTTTPTT, STTTT PTSTTTPTTITT and TTPTTP, which repeated 15, 5, 5 and 4 times, respectively. The periodic acid-Schiff (PAS) stained SDS-PAGE analysis showed that the two red glycoprotein bands accorded with the 160kDa and 80kDa protein bands of the PM proteins from the Holotrichia oblita larvae by the silver-stained SDS-PAGE analysis. Western blot analysis showed that the bigger molecular weight PM protein band disappeared after the treatment of the PM from the Holotrichia oblita larvae with the enhancin isolated from the Trichoplusia ni granulosis virus (TnGV), and some smaller molecular weight PM proteins increased correspondingly.