Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.
Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.
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RNA干扰揭示昆虫可溶性和膜结合海藻糖酶基因在几丁质生物合成中的不同功能
DOI：
10.1371/journal.pone.0010133
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发表时间：
2010-04-12
期刊：
影响因子：
3.7
通讯作者：
Zhang W
中科院分区：
文献类型：
--
作者：
Chen J;Tang B;Chen H;Yao Q;Huang X;Chen J;Zhang D;Zhang W
海藻糖酶是一种水解海藻糖产生两个葡萄糖分子的酶，在多种生理过程中起着关键作用。近年来，海藻糖酶蛋白已从几种昆虫中纯化出来，并分为可溶性（Tre-1）和膜结合（Tre-2）海藻糖酶。然而，这两种海藻糖酶在昆虫几丁质生物合成中的功能还未见报道。主要结果甜菜夜蛾（SeTre-2）的膜结合海藻糖酶的特点是在我们的实验室以前。本研究克隆了可溶性海藻糖酶基因（SeTre-1），并对这两个基因的组织分布和发育表达模式进行了研究。SeTre-1在角质层和马氏管中高表达，SeTre-2在气管和脂肪体中表达。在中肠中，两种海藻糖酶基因在不同的位置表达。此外，这两种海藻糖酶mRNA的表达谱及其酶活性表明，它们可能在几丁质生物合成中发挥不同的作用。SeTre-1或SeTre-2的RNA干扰（RNAi）是基因特异性的和有效的，在注射后72 h的效率高达83%。SeTre-1和SeTre-2的RNAi后，幼虫-蛹期和蛹-成虫期的死亡率显著提高，并对致死表型进行了分类和分析。此外，海藻糖和葡萄糖浓度的变化趋势在RNAi突变体中呈现出相反的趋势。此外，SeTre-1基因的敲除在很大程度上抑制了几丁质合成酶基因A（CHSA）的表达，并降低了角质层中的几丁质含量相对于对照昆虫的三分之二。几丁质合成酶基因B（CHSB）的表达受到SeTre-2双链RNA的抑制，中肠几丁质含量下降约25%。结论SeTre-1在表皮CHSA表达和几丁质合成中起重要作用，SeTre-2在中肠CHSB表达和几丁质合成中起重要作用。
Background Trehalase, an enzyme that hydrolyzes trehalose to yield two glucose molecules, plays a pivotal role in various physiological processes. In recent years, trehalase proteins have been purified from several insect species and are divided into soluble (Tre-1) and membrane-bound (Tre-2) trehalases. However, no functions of the two trehalases in chitin biosynthesis in insects have yet been reported. Principal Findings The membrane-bound trehalase of Spodoptera exigua (SeTre-2) was characterized in our laboratory previously. In this study, we cloned the soluble trehalase gene (SeTre-1) and investigated the tissue distribution and developmental expression pattern of the two trehalase genes. SeTre-1 was expressed highly in cuticle and Malpighian tubules, while SeTre-2 was expressed in tracheae and fat body. In the midgut, the two trehalase genes were expressed in different locations. Additionally, the expression profiles of both trehalase mRNAs and their enzyme activities suggest that they may play different roles in chitin biosynthesis. The RNA interference (RNAi) of either SeTre-1 or SeTre-2 was gene-specific and effective, with efficiency rates up to 83% at 72 h post injection. After RNAi of SeTre-1 and SeTre-2, significant higher mortality rates were observed during the larva-pupa stage and pupa-adult stage, and the lethal phenotypes were classified and analyzed. Additionally, the change trends of concentration of trehalose and glucose appeared reciprocally in RNAi-mutants. Moreover, knockdown of SeTre-1 gene largely inhibited the expression of chitin synthase gene A (CHSA) and reduced the chitin content in the cuticle to two-thirds relative to the control insects. The chitin synthase gene B (CHSB) expression, however, was inhibited more by the injection of dsRNA for SeTre-2, and the chitin content in the midgut decreased by about 25%. Conclusions SeTre-1 plays a major role in CHSA expression and chitin synthesis in the cuticle, and SeTre-2 has an important role in CHSB expression and chitin synthesis in the midgut.
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