梨小食心虫几丁质合成酶1基因的克隆与表达分析

Cloning and expression profiling of chitin synthase 1 gene in the oriental fruit moth,Grapholitha molesta( Lepidoptera: Tortricidae)

【目的】克隆梨小食心虫Grapholitha molesta(Busck)几丁质合成酶1基因,分析该基因的分子特征及时空表达模式,为探析其生理功能奠定基础。【方法】利用简并引物和RACE技术从梨小食心虫5龄幼虫和蛹中克隆几丁质合成酶1基因的全长c DNA序列,同时获得其两个可变剪切外显子序列,并用邻接法(neighbor-joining method)与其他昆虫同源序列构建系统进化树。利用RTq PCR技术研究该基因及两个可变剪切外显子在1日龄预蛹不同组织(头、体壁、脂肪体、中肠、气管和马氏管)中以及不同发育阶段(2-5龄幼虫、预蛹、蛹和成虫)的表达特性。【结果】克隆获得梨小食心虫几丁质合成酶1基因,将其命名为Gm CHS1,该基因编码1 565个氨基酸,包含了16个跨膜螺旋,两个可变剪切外显子包含177个碱基,编码59个氨基酸序列,分别命名为Gm CHS1a(Gen Bank登录号:MF000781)和Gm CHS1b(Gen Bank登录号:MF000782)。系统发育树同源分析结果表明,Gm CHS1属于几丁质合成酶1,Gm CHS1a和Gm CHS1b分别归属于可变剪切外显子CHS1a和CHS1b。组织表达模式表明,Gm CHS1基因在体壁中表达量最高,其次是在头和气管中,其余组织中表达量较低或不表达;发育表达模式表明,该基因在各个发育阶段均有表达,幼虫蜕皮期、预蛹-蛹和蛹-成虫转变过程中表达量上调。Gm CHS1a在体壁和头部的表达量高于Gm CHS1b,而在气管和脂肪体中的表达量略低于Gm CHS1b,两者在中肠和马氏管中表达量都很低。在梨小食心虫不同发育阶段,Gm CHS1a的表达趋势表现为在幼虫蜕皮和预蛹-蛹转变过程中高表达;Gm CHS1b在幼虫各个阶段表达量都较低,在预蛹-蛹和蛹-成虫转变过程中高表达。【结论】Gm CHS1a和Gm CHS1b属于昆虫几丁质合成酶1家族,其基因在梨小食心虫不同组织及发育阶段的表达量显著不同,推测其在梨小食心虫发育过程中发挥着不同的作用。本研究为进一步探索该基因在梨小食心虫体内的功能奠定了基础。

[ Aim] To clone a chitin synthase 1 gene from the oriental fruit moth, Grapholitha molesta （Busck） and to analyze its mRNA molecular characteristics and spatio-temporal expression patterns, so as to develop a basis for further research of the biological function of this gene. [ Methods ] The complete cDNA of a chitin synthase 1 gene was cloned from the 5th instar larvae and pupae of G. molesta by using degenerate primers and RACE technology, and two alternative splicing exons were obtained. Phylogenetic trees were constructed with the homologous sequences of other insects by using neighbor-joining method. The expression patterns of this gene in different tissues of 1 day-old prepupae （head, integument, fat body, midgut, trachea and Malpighian tubules） and different developmental stages （2nd -5th instar larva, prepupa, pupa and adult） were detected by RT-qPCR. [ Results ] The full-length cDNA of a chitin synthase 1 gene was cloned from G. molesta, and named GmCHS1. It encodes 1 565 amino acids, and contains 16 transmembrane helices. The two alternative splicing exons are located in the region consisting of 177 nucleotides that encodes 59 amino acid residues, and named GmCHSla （GenBank accession no.： MF000781） and GmCHSlb （GenBank accession no.： MF000782）, respectively. The phylogenetic trees showed that GmCHS1 belongs to the chitin synthase 1 family, and the two alternative exons of GmCHSla and GmCHSlb are grouped into the corresponding two classes, CHSla and CHSlb. The tissue expression profiles of GmCHS1 indicated that it had the highest expression level in the integument, followed by trachea and head, while had a low expression level or was hardly expressed in other tissues. The developmental expression profiles of GmCHS1 showed that it was expressed throughout the whole developmental stages, and showed high expression during larval-larval molting, prepupal-pupal and pupal-adult transformation. The expression levels of GmCHSla were higher in the integument and head but slightly lower in trachea and fat body than those of GmCHS1 b. During growth and development of G. molesta, GmCHSla was mainly expressed during larval-larval molting and prepupal-pupal transformation, while GmCHSlb was expressed during the prepupal-pupal and pupal-aduh transformation. [Conclusion] GmCHSla and GmCHSlb should be classified into chitin synthase 1 family. The expression levels of GmCHSla and GmCHSlb are significantly different in different tissues and developmental stages of G. molesta, suggesting that these two genes play different roles in the growth and development of G. molesta. This study lays the foundation for the further research of the function of GmCHSla and GmCHSlb in G. molesta.