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【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。
【Objective】 The objective of this study was to clone and analyze the coding sequence of the Helicoverpa armigera HaTO-like gene in cotton bollworm (Helicoverpa armigera). The temporal and spatial expression profiles of the gene and the transcriptional changes after HaSNPV infection of Helicoverpa armigera were detected. Function to provide a theoretical basis. 【Method】 The full-length cDNA sequence of HaTO-like gene was cloned by RT-PCR for the first time. The nucleotide sequence and amino acid sequence of HaTO-like gene were analyzed by several bioinformatics softwares. Fluorescence quantitative PCR technique was used to detect the expression of HaTO-like gene in larval tissues and adult tissues at different developmental stages of Helicoverpa armigera and HaSNPV infection. 【Result】 The full-length cDNA of HaTO-like gene was 994 bp in length and 756 bp in open reading frame (ORF) encoding a polypeptide of 251 amino acids with a 23 amino acid signal peptide at the N terminus. Further sequence analysis showed that the amino acid sequence identity of the Helicoverpa armigera HaTO-like with other insect homologous proteins was not very high, ranging from 39% to 61%. The most closely related phylogenetic tree was found between silkworm and Ostrinia. Quantitative real-time PCR results showed that the expression level of this gene was relatively high at 0 h and 0 d of the first instar in cotton bollworm and higher in larval head and epidermis than that of other larvae. Ministry and foot expression is also relatively high. The virus infection significantly induced the expression of the gene in the head and epidermis of H. armigera larvae. 【Discussion】 This study cloned the full-length cDNA sequence of HaTO-like gene from Helicoverpa armigera, analyzed the sequence characteristics and expression profile of HaTO-like gene, and laid the theoretical foundation for further elucidating the function of this gene.