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【目的】海藻糖酶(trehalase,Tre)是昆虫体内海藻糖代谢的一个关键酶,包括可溶型(Tre1)和膜结合型(Tre2)两种类型,在昆虫发育和能量调节中具有重要作用。本研究旨在克隆稻纵卷叶螟Cnaphalocrocis medinalis海藻糖酶基因(Cm Tre),解析其在稻纵卷叶螟不同发育阶段和不同组织中的表达模式,分析该基因及酶蛋白的分子特征。【方法】通过稻纵卷叶螟转录组数据结合RACE技术,克隆稻纵卷叶螟Cm Tre的全长c DNA序列,并对该基因进行生物信息学分析;采用实时荧光定量PCR(RT-q PCR)解析Cm Tre在稻纵卷叶螟不同发育阶段及成虫不同组织部位的m RNA表达模式。【结果】获得两种类型的稻纵卷叶螟Cm Tre基因,即可溶型海藻糖酶基因Cm Tre1和膜结合型海藻糖酶基因Cm Tre2。Cm Tre1的全长c DNA长度为2 364 bp,开放阅读框长1 704 bp,编码567个氨基酸;Cm Tre2的全长c DNA长度为2 079 bp,开放阅读框长1 923 bp,编码640个氨基酸。生物信息学分析表明,Cm Tre前端有一个信号肽,Cm Tre1无跨膜结构,Cm Tre2有一个跨膜结构。同源性和聚类分析表明,Cm Tre1和Cm Tre2的氨基酸序列与竹蠹螟Omphisa fuscidentalis海藻糖酶Tre1和Tre2氨基酸序列的一致性最高,分别为74%和79%。同源建模预测结果显示,Cm Tre1的三维分子结构包含19个α螺旋和2个β折叠片;Cm Tre2的三维分子结构含有23个α螺旋,没有β折叠片。RT-q PCR结果显示,Cm Tre在稻纵卷叶螟整个发育历期都有表达,在成虫期表达水平最高,在整个幼虫期都有相对稳定的表达;Cm Tre1在蛹期表达水平最低,Cm Tre2在5龄幼虫时期表达水平最低。Cm Tre在所检测的成虫组织(中肠、体壁、马氏管、头、卵巢、脂肪体、肌肉和精巢)中均有表达;Cm Tre1在中肠和体壁中的表达水平较高,Cm Tre2在肌肉和中肠中的表达水平较高。【结论】本研究克隆了稻纵卷叶螟两种类型的海藻糖酶基因,分析了其基因特征和表达模式。研究结果为阐明海藻糖酶基因的功能进而以海藻糖酶基因为靶标防治害虫奠定了基础。
【Objective】 Trehalase (Tre) is a key enzyme in the metabolism of trehalose in insects, including Tre1 and Tre2, which play an important role in insect development and energy regulation . The aim of this study was to clone the Cmn of Cnaphalocrocis medinalis and to analyze its expression pattern in different developmental stages and different tissues of C. medinalis, and to analyze its molecular characteristics. 【Method】 The full-length c DNA sequence of CmTr was cloned by RT-qRT and RACE techniques. The gene was analyzed by bioinformatics analysis. Real-time PCR (RT-q PCR) was used to analyze the m RNA expression pattern of Cm Tre at different developmental stages of rice leaf roller and adult tissues. 【Result】 Two types of Cm Tre gene of rice leaf roller were obtained, namely soluble trehalase gene Cm Tre1 and membrane bound trehalase gene Cm Tre2. The length of full length c DNA of Cm Tre1 was 2 364 bp and the open reading frame was 1 704 bp, encoding 567 amino acids. The full length c DNA of Cm Tre2 was 2 079 bp with an open reading frame of 1 923 bp encoding 640 Amino acids. Bioinformatics analysis showed that there was a signal peptide at the front of Cm Tre, no transmembrane structure at Cm Tre1, and a transmembrane structure at Cm Tre2. Homology and cluster analysis showed that the amino acid sequences of Cm Tre1 and Cm Tre2 had the highest identities with those of Omphisa fuscidentalis trehalase Tre1 and Tre2, which were 74% and 79%, respectively. The results of homology modeling showed that the three-dimensional molecular structure of Cm Tre1 contains 19 α-helices and two β-sheets. The three-dimensional molecular structure of Cm Tre2 contains 23 α-helices without β-sheet. The results of RT-q PCR showed that Cm Tre was expressed throughout the developmental stages of C. medinalis, and had the highest expression level at adult stage and relatively stable throughout larval stage. Cm Tre1 had the lowest expression level at pupal stage, Cm Tre2 had the lowest expression level in the 5th instar larvae. Cm Tre was detected in all adult tissues (midgut, body wall, Markovs, head, ovary, fat body, muscle and testis); Cm Tre1 was highly expressed in the midgut and the body wall, Cm Tre2 is highly expressed in muscle and midgut. 【Conclusion】 In this study, two types of trehalase gene of rice leaf roller were cloned and their gene characteristics and expression patterns were analyzed. The results laid the foundation for the elucidation of the function of trehalase gene and the control of pests by the trehalase gene.