关键词: 稻纵卷叶螟, 海藻糖酶, 基因克隆, 序列分析, 表达模式

Abstract: 【Aim】 Trehalase (Tre) is a key enzyme in trehalose metabolism of insects and plays important roles in the development and regulation of energy. Insects possess two types of trehalases, i.e., soluble trehalase (Tre1) and membranebound trehalase (Tre2). This study aims to clone trehalase gene (CmTre) from the rice leaf folder, Cnaphalocrocis medinalis, to clarify its expression patterns in different tissues and developmental stages, and to analyze the molecular characteristics of the two types of the gene and their products. 【Methods】 Based on the transcriptome data of C. medinalis, the full-length cDNA of CmTre was cloned using the rapid amplification of cDNA ends (RACE)-PCR and analyzed by bioinformatics. CmTre mRNA expression levels in different tissues of adults and developmental stages of C. medinalis were detected by using real-time quantitative PCR (RT-qPCR). 【Results】 We cloned two types of CmTre, i.e., soluble trehalase gene CmTre1 and membrane-bound trehalase gene CmTre2. The full-length cDNA of CmTre1 is 2 364 bp containing a 1 704 bp open reading frame (ORF) that encodes 567 amino acids, while that of CmTre2 is 2 079 bp containing a 1 923 bp ORF that encodes 640 amino acids. Bioinformatics analysis indicated that CmTre includes a signal peptide and CmTre1 has no transmembrane domain, whereas CmTre2 contains a transmembrane domain. Homology and phylogenetic analyses showed that the amino acid sequences of CmTre1 and CmTre2 have the highest identities with those of Tre1 and Tre2 from Omphisa fuscidentalis, which are 74% and 79%, respectively. Homology modeling analysis demonstrated that the tertiary structure of CmTre1 is composed of 19 α-helices and 2 β-sheets while that of CmTre2 is composed of 23 α-helices and without β-sheet. RT-qPCR revealed that CmTre was expressed throughout all developmental stages of C. medinalis, with the highest expression level in adult and a relatively stable expression level in larval stage. CmTre1 was expressed at the lowest level in pupa, while CmTre2 was expressed at the lowest level in the 5th instar larva. CmTre was expressed in all the adult tissues tested (midgut, integument, malpighian tubules, head, ovary, fat body, muscle and testis). CmTre1 was expressed at higher levels in the midgut and integument while CmTre2 was expressed at higher levels in the muscle and midgut. 【Conclusion】 In this study, genes of a soluble and a membrane-bound form of trehalase in C. medinalis were cloned, and their characteristics and expression patterns were analyzed. The findings lay the foundation for further research on the functions of trehalase genes and then using the genes as the targets of pest control.

Key words: Cnaphalocrocis medinalis, trehalase, gene cloning, sequence analysis, expression pattern