Cloning, expression and chitin-binding activity of two peritrophin-like protein genes in the common cutworm, Spodoptera litura
Cloning, expression and chitin-binding activity of two peritrophin-like protein genes in the common cutworm, Spodoptera litura
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斜纹夜蛾两种围营养蛋白样蛋白基因的克隆、表达及几丁质结合活性
DOI：
10.1111/1744-7917.12055
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发表时间：
2014-08-01
期刊：
影响因子：
4
通讯作者：
Feng, Qi-Li
中科院分区：
文献类型：
--
作者：
Chen, Wei-Jun;Huang, Li-Xia;Feng, Qi-Li
昆虫中肠分泌半透性围食膜（PM），在保护中肠和帮助食物消化方面起着重要作用。鳞翅目幼虫产生1型PM，当昆虫发育到变态阶段时，该PM被降解。然而，其潜在机制尚不清楚。在本研究中，两个peritrophin样蛋白（peritrophin-57和37）的中肠表达序列标签库和转录组中的常见的地老虎，斜纹夜蛾。根据其共同序列区，通过实时定量聚合酶链反应检测了时间和空间表达模式以及对20-羟基蜕皮激素（20 E）诱导和饥饿的反应。还使用竞争对手calcofluor研究了几丁质结合活性。开放阅读框为1554和1020 bp。它们共有四个高度保守的围肌肽A结构域，仅在中肠表达，而在脂肪体、表皮、马氏管和血淋巴等其他组织中不表达。它们的转录表达仅在幼虫期检测到，而在卵、预蛹、蛹和成虫中检测不到。纯化的peritrophin-37蛋白与几丁质的结合呈剂量依赖性。这些结果表明，这两种蛋白是围肌肽，PM的结构组成部分。此外，两种围肌肽的信使RNA水平显着下调20 E注射，而喂养/饥饿的表达没有影响。这些结果表明，20 E滴度的增加可能是控制变态过程中PM降解的重要因素。
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 expressed 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 of 20E titer may be an important factor which controls the degradation of PM during metamorphosis.