Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was iso...Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761展开更多
The peritrophic matrix （PM） is essential for insect digestive system physiol- ogy as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for de...The peritrophic matrix （PM） is essential for insect digestive system physiol- ogy as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata （Lepidoptera： Noctuidae）, a major pest of cruciferous oilseed crops in North America. Liquid chromatography-tandem mass spectrometry analyses of the PM identified 82 proteins classified as： （i） peritrophins, including a new class with a CBDIII domain; （ii） enzymes involved in chitin modification （chitin deacetylases）, di- gestion （serine proteases, aminopeptidases, carboxypeptidases, lipases and ^-amylase） or other reactions （/^-l,3-glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase）; （iii） a heterogenous group consisting of polycalin, REPATs, serpin, C-Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut, cDNAs encoding chitin synthase-2 （McCHS-2）, chitinase （McCHI）, and fl-N-acetylglucosaminidase （McNAG） enzymes, in- volved in PM chitin metabolism, were also identified. McCHS-2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS-2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.展开更多
基金Supported by Natural Science Foundation of Heilongjiang Province (C2007-7)Scientific and Technical Innovation Fund of Harbin (RC2006QN002027)Northeast Agricultural University Research Fund (2005)
文摘Chitin is the most widespread amino polysaccharide in nature. Chitin synthase （CHS） plays an important role in chitin formation in the cuticle and the peritrophic membrane （PM） lining the midgut. Total RNA was isolated from the cuticle of Mamestra brassicae （L.） fourth instar larva, cDNA sequence was cloned by RT-PCR and Rapid Amplification of cDNA Ends （RACE）. cDNA 5 220 bp in length, contained an open reading frame of 4 704 bp coding for a polypeptide of 1 567 amino acid residues with a predicted molecular weight of 178.3 ku and its pI was 6.42. The deduced amino acid sequence from Mi brassicae （L.） shared the high level of identity with chitin synthase sequences from other insects, especially lepidopteran insects, cDNA sequence has been deposited with GenBank under accession No. GQ281761
文摘The peritrophic matrix （PM） is essential for insect digestive system physiol- ogy as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata （Lepidoptera： Noctuidae）, a major pest of cruciferous oilseed crops in North America. Liquid chromatography-tandem mass spectrometry analyses of the PM identified 82 proteins classified as： （i） peritrophins, including a new class with a CBDIII domain; （ii） enzymes involved in chitin modification （chitin deacetylases）, di- gestion （serine proteases, aminopeptidases, carboxypeptidases, lipases and ^-amylase） or other reactions （/^-l,3-glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase）; （iii） a heterogenous group consisting of polycalin, REPATs, serpin, C-Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut, cDNAs encoding chitin synthase-2 （McCHS-2）, chitinase （McCHI）, and fl-N-acetylglucosaminidase （McNAG） enzymes, in- volved in PM chitin metabolism, were also identified. McCHS-2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS-2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.
