Isolation And Identification Of Peritrophic Membrane Target Proteins

The insect midgut epithelium is generally lined with a unique chitin/protein structure, the peritrophic membrane (PM), playing important roles in the intestinal biology of the insect. Based on the understanding/study of PM in molecular biology and bio-chemistry standard, the studies focus on molecular structure and function of the PM in recent years. Moreover, PM proteins are important determinant for the structural formation and function of insect, so we isolated and identified several proteins from the PMs of Spodoptera exigua and Helicoverpa armigera larvae, which are world widely important insect in agriculture, by cDNA cloning to develop the biocontrol associated gene for S. exigua and H. armigera.To study PM proteins, S. exigua and H. armigera larval midgut cDNA expression library was constructed respectively. The original cDNA expression library from those two insects contained 5.51×10~6 recombinants and 4.82×10~6 pfu/ml separately. And the recombination rate was 99.97% and 99.79% each. The titering of amplified library is 2.41×10~9 pfu/ml and 1.5×10~9 pfu/ml. The average length of the inserted fragments was 1.8 kb and 2.0 kb by PCR analysis. The construction of two cDNA libraries with high quality lays a foundation for the future study.S. exigua PM proteins were extracted then the antiserum against S. exigua PM proteins was prepared successfully. Protein composition was analyzed by PAS staining method and the results showed that PM has a high glycoprotein content, however, glycoproteins are low level in the midgut epithelium from both S. exigua and H. armigera.S.exigua midgut cDNA expression library was screened for cDNAs coding for PM proteins by immune-screening with different antiserum. 374 of positive clones were got. Among of them, the sequence similarity of SeIIM-6/8/20 resulted in ifentification of Mamestra configurata IIM. But they were lack of 5'-end sequence. SeIIM-6/20 encoded 3 and 4 chitin binding domains (CBDs) each but SeIIM-8 contained five CBDs and one specific aspartic acid-rich region as well as one threonine-rich mucin domain.Using the plasmid conferred to SeP159 gene (4.8kb) as template, p1226 gene which contained one signal peptide and three CBDs was amplified by PCR method. Then, p1226 was recombined to pQE30 vector and expressed.SeP30 clone harbored secbp66 gene which encodes peritrophin (Genbank accession number is EU139126). The cDNA was 2,169bp, and the longest open reading frame coded for 602 amino acids. The similarity to M.configurata peritrophin1 was 48% in amino acids sequence. It contained a signal peptide composition of 17 amino acids and 7CBDs. SeCBP66 was shown as a 95kDa protein by SDS-PAGE analysis. SeCBP66 had chitin binding activity and was strongly associated with PM, which is similar to the currently known peritrophin type PM proteins. To study the function of single CBD, cbd1 gene coding for CBD1 which contained one signal peptide and one CBD domain was amplified by PCR from secbp66 gene. CBD1 was expressed in E.coli and insect cells by spot blot analysis.Carboxylesterase gene sec1 was isolated from S. exgiua library with IIM-specific polyclonal antiserum from T. ni. The GenBank accession number is EF580101. It was expressed in E.coli and insect cells respectively. The activity for Sec1 was 1.3 nmol/100μl enzyme buffer with the a-NA as substrate.H. armigera intestinal mucin gene hat12, hat15, hat46 (haiim-46) and haiim-86 were cloned from H.armigera cDNA library with total PM polyclonal antiserum from S.exigua. The former three genes were 1772bp, 1728 bp and 2857bp in size; haiim-46 gene encoded HaIIM-46 which owned 3 mucin domains and 8 CBDs (partial in 5' ends). The cDNA of HaIIM86 was 3,147bp, and the longest open reading frame was 2,517bp. The accession number in GenBank for HaIIM86 was EU047712. HaIIM86 contained one signal peptide, five chitin binding domains, one mucin domain and two glycine-aspartic acid (D-G) rich region. These two D-G rich regions which was reported firstly in insect protein located between the third to the fifth CBD. The former region contained one repeating unit, CDGSCPDNGGNDGN repeated 2 times and the later, NDGG repeating unit repeats 20 times. Haiim86 was cloned into pQE30 vector and 200kDa protein was detected in E.coli M15 by Western blot analysis. Moreover, HaIIM86 was expressed in insect cells and the function study on HaIIM86 demonstrated that it had chitin binding activity and could be degraded by HaGV Enhancin. The identification of HaIIM86 expanded knowledge of the insect PM, which may prove to be a prime target for novel insect control strategies.