Previously, we have reported that a gene encoding Bombyxmoriβ-N-acetylglucosaminidase 2 (BmGlcNAcase2) has been identified differentially expressed in the midgut of Bombyxmori strain NB resistant to nucleopolyhedrovi...Previously, we have reported that a gene encoding Bombyxmoriβ-N-acetylglucosaminidase 2 (BmGlcNAcase2) has been identified differentially expressed in the midgut of Bombyxmori strain NB resistant to nucleopolyhedrovirus (BmNPV), strain 306 susceptible to NPV and a near isogenic line BC9 with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). To get more knowledge about the relationship between β-N-acetylglucosaminidase and the resistance of NPV, in this study, the 1542 bp open reading frame of a putative bombyxmoriβ-N-acetylglucosaminidase 2 gene (BmGlcNAcase2) was amplified from a pool of bombyxmoricDNAs and inserted into the prokaryotic expression plasmid pET-30a(+).Western blotting analysis showed that BmGlcNAcase2 was expressed in hemolymph, ovary, testis, fat body, trachea, midgut and silk gland of fifth instar larvae respectively . Immunofluoresence analysis indicated that BmGlcNAcase2 was mainly located to the cytoplasm or some structure in cytoplasm.展开更多
β-N-acetylglucosaminidases are crucial enzymes involved in chitin degrada- tion in insects. We identified a β-N-acetylglucosaminidase gene （LmNAG1） from Locusta migratoria. The full-length complementary DNA （cDNA...β-N-acetylglucosaminidases are crucial enzymes involved in chitin degrada- tion in insects. We identified a β-N-acetylglucosaminidase gene （LmNAG1） from Locusta migratoria. The full-length complementary DNA （cDNA） of LmNAG1 consists of 2 667 nucleotides, including an open reading frame （ORF） of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5＇- and 3＇- ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized β-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real- time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA （dsRNA）. As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAGl-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process ofL. migratoria.展开更多
文摘Previously, we have reported that a gene encoding Bombyxmoriβ-N-acetylglucosaminidase 2 (BmGlcNAcase2) has been identified differentially expressed in the midgut of Bombyxmori strain NB resistant to nucleopolyhedrovirus (BmNPV), strain 306 susceptible to NPV and a near isogenic line BC9 with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). To get more knowledge about the relationship between β-N-acetylglucosaminidase and the resistance of NPV, in this study, the 1542 bp open reading frame of a putative bombyxmoriβ-N-acetylglucosaminidase 2 gene (BmGlcNAcase2) was amplified from a pool of bombyxmoricDNAs and inserted into the prokaryotic expression plasmid pET-30a(+).Western blotting analysis showed that BmGlcNAcase2 was expressed in hemolymph, ovary, testis, fat body, trachea, midgut and silk gland of fifth instar larvae respectively . Immunofluoresence analysis indicated that BmGlcNAcase2 was mainly located to the cytoplasm or some structure in cytoplasm.
文摘β-N-acetylglucosaminidases are crucial enzymes involved in chitin degrada- tion in insects. We identified a β-N-acetylglucosaminidase gene （LmNAG1） from Locusta migratoria. The full-length complementary DNA （cDNA） of LmNAG1 consists of 2 667 nucleotides, including an open reading frame （ORF） of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5＇- and 3＇- ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized β-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real- time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA （dsRNA）. As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAGl-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process ofL. migratoria.
