棉铃虫HaTO-like基因的克隆、序列分析和表达特征

Cloning,sequence analysis and expression of a takeout-like gene in Helicoverpa armigera

【目的】克隆和分析了棉铃虫Helicoverpa armigera HaTO-like基因的编码框序列,检测了该基因的时空表达谱以及在棉铃虫感染核型多角体病毒HaSNPV后的转录变化,为深入研究该基因的功能提供理论依据。【方法】本研究利用RT-PCR的方法首次克隆获得HaTO-like基因的全长cDNA序列,通过几种生物信息学软件对该基因的核苷酸序列和氨基酸序列进行了分析,并利用荧光定量PCR技术检测了该基因在棉铃虫不同发育阶段、幼虫组织和成虫组织的表达情况,以及HaSNPV感染对HaTO-like基因表达的影响。【结果】棉铃虫HaTO-like基因cDNA全长为994 bp,开放阅读框为756 bp,编码251个氨基酸,其蛋白序列的N端含有23个氨基酸的信号肽。进一步的序列分析表明棉铃虫HaTO-like与其他昆虫同源蛋白的氨基酸序列一致性不是太高,大概在39%~61%之间,其中与家蚕和脐橙螟在系统进化上关系最近。荧光定量PCR结果表明该基因在棉铃虫的5龄0 h和成虫第1天的的表达量相对较高,在幼虫的头部和表皮内的表达量较其他幼虫组织较高,在成虫的头部和足的表达量也相对较高。而病毒感染则显著地诱导了该基因在棉铃虫幼虫头部和表皮内的表达。【讨论】本研究克隆了棉铃虫HaTO-like基因的全长cDNA序列,分析了该基因的序列特征和表达谱,为进一步阐释该基因的功能奠定理论基础。

[Objectives] To interpret the function of a HaTO-like gene in Helicoverpa armigera, we cloned the full-length cDNA of this gene, determined its expression profile in different developmental stages and different tissues, and detected its transcripts in the head and epidermis of larvae after HaSNPV infection. [Methods] Full-length HaTO-like cDNA was amplified by RT-PCR and the nucleotide and amino acid sequences of the gene were further analyzed using bioinformatics software. qRT-PCR was also used to determine the spatio-temporal expression patterns of the gene and its transcript after HaSNPV infection. [Results] Sequence analysis showed that the full-length cDNA of the HaTO-like gene was 994 bp, which includes a 756 bp ORF encoding region comprised of 251 amino acids, and that it shares relative low sequence identity with homologous proteins of other insects. The results of q RT-PCR indicated that the HaTO-like gene was more highly expressed in fifth instar larvae at 0 h, and in one day old adults, than in other stages. In larvae, the HaTO-like gene was mainly expressed in the head and epidermis whereas in adults it was most highly expressed in the legs. The expression of the HaTO-like gene was also induced in head and epidermis of H. armigera after HaSNPV infection. [Conclusion] This study determined the sequence characteristics of the HaTO-like gene, and its expression profiles and establishes a theoretical foundation for further elucidating the functions of the HaTO-like gene in H. armigera.