关键词: 梨小食心虫, 几丁质合成酶, 表达谱, RT-qPCR, 可变剪切外显子

Abstract: 【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 GmCHS1a (GenBank accession no.: MF000781) and GmCHS1b (GenBank accession no.: MF000782), respectively. The phylogenetic trees showed that GmCHS1 belongs to the chitin synthase 1 family, and the two alternative exons of GmCHS1a and GmCHS1b are grouped into the corresponding two classes, CHS1a and CHS1b. 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 GmCHS1a were higher in the integument and head but slightly lower in trachea and fat body than those of GmCHS1b. During growth and development of G. molesta, GmCHS1a was mainly expressed during larval-larval molting and prepupal-pupal transformation, while GmCHS1b was expressed during the prepupal-pupal and pupal-adult transformation. 【Conclusion】 GmCHS1a and GmCHS1b should be classified into chitin synthase 1 family. The expression levels of GmCHS1a and GmCHS1b 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 GmCHS1a and GmCHS1b in G. molesta.  

Key words: Grapholitha molesta; chitin synthase, expression profile, RT-qPCR, alternative exons