黏虫咽侧体抑制神经肽基因克隆与成虫期表达模式分析

Cloning and expression analysis of allatostatin gene in adults of the oriental armyworm,Mythimna separata(Walker)

为明确黏虫咽侧体抑制神经肽基因(allatostatin,AST)在调控黏虫Mythimna separata(Walker)保幼激素(juvenile hormone,JH)中的功能,采用RT-PCR和RACE技术克隆获得的黏虫AST基因cDNA全长序列902bp,开放阅读框378bp,编码125个氨基酸。蛋白分子量为14.33kD,等电点为9.25,具有C型AST典型的PISCF序列。进化树分析表明,黏虫AST氨基酸序列与一点黏虫、草地贪夜蛾、棉铃虫氨基酸序列相似性高达80%以上。实时荧光定量PCR结果表明AST在成虫期的表达有明显组织和时间特异性。AST在飞行肌中表达量最高,头部其次,卵巢最低;黏虫头部AST表达量在7日龄最高,飞行肌中AST基因的表达量在1日龄最高。本研究对进一步揭示黏虫AST基因对JH调控作用,明确黏虫JH调控迁飞和生殖的相关的分子调控机制具有重要意义。

In order to clarify the role of the allatostatin gene（AST）in the process of juvenile hormone synthesis and release of Mythimna separata（Walker）,the AST gene of M.separata was cloned by RT-PCR and RACE.The full length of cDNA was 902 bp,which contained a 378 bp open reading frame（ORF）encoding 125 amino acid residues with a molecular mass of 14.33 kD and a theoretical isoelectric point of 9.25.The typical structure of C-type AST（PISCF）was located in the deduced amino acid sequence.Phylogenetic analysis indicated that AST protein of M.separata had a high similarity（up to 80%）to those of Pseudaletia unipuncta,Spodoptera frugiperda and Helicoverpa armigera.The qRT-PCR analysis showed that the expression levels of AST gene were significantly different at different stages and in various tissues of the female M.separata,mostly expressed in flight muscle and head,weakly expressed in the ovary.The expression was the highest in the head at 7 dbut in flight muscle at 1 d.This study has a significance in enriching our knowledge about the function of AST on JH and the molecular mechanism of JH during flight and reproduction in M.separata.