Insect midgut secretes a semi-permeable peritrophic membrane （PM）, which plays important roles in protecting the midgut and helping with food digestion. The lep- idopteran larvae produce type 1 PM, which is degraded...Insect midgut secretes a semi-permeable peritrophic membrane （PM）, which plays important roles in protecting the midgut and helping with food digestion. The lep- idopteran larvae produce type 1 PM, which is degraded when insects develop into the metamorphic stages. However, the underlying mechanism is unclear. In the present study, two peritrophin-like proteins （peritrophin-57 and 37） were identified from the midgut expression sequence tag library and transcriptome of the common cutworm, Spodoptera litura. The temporal and spatial expression patterns and responses to the induction of 20- hydroxyecdysone （20E） and starvation were examined by real-time quantitative polymerase chain reaction according to their common sequence region. The chitin-binding activity was also studied using a competitor, calcofluor. The open reading frames are 1 554 and 1 020 bp, respectively. They shared four highly conserved peritrophin-A domains and were ex- pressed only in the midgut rather than in the other tissues, including fat body, epidermis, Malpighian tube and hemolymph. Their transcriptional expression could only be detected at the larval stages rather than in eggs, prepupae, pupae and adults. The purified protein of peritrophin-37 bound to chitin in a dose-dependent manner. These results indicate that the two proteins are peritrophins, the structural components of PM. In addition, the messenger RNA levels of the two peritrophins were significantly down-regulated by 20E injection, whereas feeding/starvation had no effect on the expression. These findings suggest that the increase of20E titer may be an important factor which controls the degradation of PM during metamorphosis.展开更多
The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix （PM）, composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our underst...The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix （PM）, composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins （insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1） and the chitin biosynthetic or modifying enzymes （chitin synthase-B and chitin deacetylase 1） in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing （by 24 h） for the gene encoding chitin deacetylase 1 and a slower rate of silencing （by 72 h） for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.展开更多
基金This research was supported by grants from the National Basic Research Program of China （973 Program, no. 2012CB 114101）, National Natural Science Foundation of China （grant nos 31172158 and 30900152） and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
文摘Insect midgut secretes a semi-permeable peritrophic membrane （PM）, which plays important roles in protecting the midgut and helping with food digestion. The lep- idopteran larvae produce type 1 PM, which is degraded when insects develop into the metamorphic stages. However, the underlying mechanism is unclear. In the present study, two peritrophin-like proteins （peritrophin-57 and 37） were identified from the midgut expression sequence tag library and transcriptome of the common cutworm, Spodoptera litura. The temporal and spatial expression patterns and responses to the induction of 20- hydroxyecdysone （20E） and starvation were examined by real-time quantitative polymerase chain reaction according to their common sequence region. The chitin-binding activity was also studied using a competitor, calcofluor. The open reading frames are 1 554 and 1 020 bp, respectively. They shared four highly conserved peritrophin-A domains and were ex- pressed only in the midgut rather than in the other tissues, including fat body, epidermis, Malpighian tube and hemolymph. Their transcriptional expression could only be detected at the larval stages rather than in eggs, prepupae, pupae and adults. The purified protein of peritrophin-37 bound to chitin in a dose-dependent manner. These results indicate that the two proteins are peritrophins, the structural components of PM. In addition, the messenger RNA levels of the two peritrophins were significantly down-regulated by 20E injection, whereas feeding/starvation had no effect on the expression. These findings suggest that the increase of20E titer may be an important factor which controls the degradation of PM during metamorphosis.
文摘The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix （PM）, composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins （insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1） and the chitin biosynthetic or modifying enzymes （chitin synthase-B and chitin deacetylase 1） in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing （by 24 h） for the gene encoding chitin deacetylase 1 and a slower rate of silencing （by 72 h） for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.
