小菜蛾几丁质酶基因的克隆及表达分析

Cloning and expression profiling of a Chitinase gene from Plutella xylostella

昆虫几丁质酶参与昆虫蜕皮、消化、防御及免疫等多种生理过程,在昆虫的变态和发育中发挥着重要作用。本研究采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆获得小菜蛾Plutella xylostella几丁质酶基因,命名为PxyChi。该基因开放阅读框长1677 bp,编码558个氨基酸,推测的蛋白分子量为62.03 kDa。氨基酸序列分析表明,小菜蛾几丁质酶第1-19位氨基酸为蛋白的信号肽,且该序列具有典型昆虫几丁质酶的4个保守氨基酸序列。蛋白结构域分析表明,PxyChi属于几丁质酶Group II家族。进化树分析表明:PxyChi与同属鳞翅目二化螟Chilo suppressalis的几丁质酶亲缘关系最近,为75.5%。不同发育时期的荧光定量PCR分析表明,PxyChi在小菜蛾各个发育时期的表达量不同,其中PxyChi在2、3、4龄幼虫、蛹和成虫中的表达量分别是1龄幼虫的28.22、0.76、0.50、84.83和286倍,PxyChi在成虫的表达量最高。暗示PxyChi蛋白可能参与小菜蛾蛹期旧表皮降解和成虫翅的发育等生理过程。本研究可为探索几丁质酶在小菜蛾发育中的功能提供理论依据。

Insect chitinase,which is involved in insect molting,digestion,defense,and immunity,plays an important role in insect metamorphosis and development.In this research,the gene coding for Chitinase gene of Plutella xylostella was cloned by Rapid amplification cDNA ends（RACE） and reverse transcription PCR（RT-PCR）,and named as PxyChi.The sequencing results showed that the full-length of the open reading frame in PxyChi was 1677 bp,encoding 558 amino acids with an estimated molecular mass of 62.03 kDa.Amino acid sequence analysis showed that the signal peptide of PxyChi locates from the 1^st to the 19^th amino acid and PxyChi contains four conservative domains of chitinase.The analysis of protein structure showed that PxyChi belonged to the chitinase Group II family.Phylogenetic tree analysis showed that PxyChi had the closest genetic relationship with Chilo suppressalis（up to 75.5%）.The relative expression analysis further indicated that PxyChi was expressed in different developmental stages with different expression level and had the highest expression level in adults.The expression level in 2^nd,3^rd,4^th instar larva,pupal,and adult was about 28.22,0.50,84.83,and 286 folds of that in 1^（st instar larva.It is suggested that PxyChi might participate in the physiological processes such as the degradation of the old epidermis and the the development of adult wing.These results could provide a theoretical basis for exploring the function of chitinase in the development of P.xylostella.