棉铃虫中HSF1的分子克隆、选择性剪接及亚细胞定位研究

Molecular characterization,alternative splicing and subcellular localization of HSF1 in the cotton bollworm,Helicoverpa armigera

昆虫滞育过程中热休克蛋白的表达调节机制尚不明确,一个重要的原因是缺乏对其转录调控因子(热休克因子HSF1)的研究。因此,本文重点研究HSF1以期为阐明滞育昆虫中的热休克蛋白的调控机制打下基础,进而为开发新型的棉铃虫防治策略提供依据。首先,从棉铃虫中克隆了HSF1基因,对棉铃虫HSF1基因序列分析及进化树构建。随后在棉铃虫不同组织中发现了HSF1的7个选择性剪接体。最后,鉴定并分析棉铃虫中的7个HSF1的选择性剪接体,并对它们的亚细胞定位进行了研究。结果表明:棉铃虫HSF1基因的全长c DNA序列全长2 275 bp,编码一个655 aa的蛋白质,预测其分子量为72. 26 k Da,命名为Har-HSF1 (Gen Bank登录号为KP307027)。根据预测的蛋白质序列进行分析,表明棉铃虫HSF1蛋白包含4个经典的功能域:DNA结合域DBD,疏水性的七肽重复域HR-A/B,疏水性的七肽重复域HR-C以及C末端反式激活结构域CTAD。这些说明HSF1蛋白是一个在生物进化上非常保守的蛋白质。此外,发现了HSF1的7个选择性剪接体,命名为HSF1-A到HSF1-G。亚细胞定位研究表明所有的选择性剪接体都定位在细胞核中,这为它们调节靶基因提供了便利。本研究可以为进一步研究滞育昆虫中热休克蛋白的分子调控机制打下了基础。

The regulatory mechanism of hsps during insect diapause is unknown,primarily because of the lack of basic information about HSF1,a key regulator of heat shock proteins.Therefore,this paper focuses on the study of HSF1 in order to clarify the regulation mechanism of heat shock proteins in diapause insects to lay the foundation,and then provide a basis for the development of a new type of pest control strategy.First,we cloned the HSF1 gene from the cotton bollworm and sequenced the HSF1 gene of cotton bollworm and constructed the phylogenetic tree.Subsequently,seven alternative splice variants of HSF1 were found in different tissues of Helicoverpa armigera.Finally,seven alternative spliced variants of HSF1 in H.armigera were identified and analyzed,and their subcellular localization was studied.The results showed that the full-length cDNA sequence of HSF1 gene of cotton bollworm was 2 275 bp in length and encoded a protein of 655 aa.The molecular weight was predicted to be 72.26 kDa and named as Har-HSF1(GenBank accession number KP307027).Based on the predicted protein sequence analysis,it was shown that the cotton bollworm HSF1 contains four classical domains:The DNA binding domain DBD,the hydrophobic heptad repeat domain HR-A/B,the hydrophobic heptad repeat domain HR-C and C Terminal transactivation domain CTAD.These show that HSF1 protein is a very conserved biological evolutionary protein.In addition,seven alternative splice variants of HSF1 were found,named HSF1-A to HSF1-G.Subcellular localization studies indicate that all alternative spliceosomes are localized in the nucleus,which facilitates their regulation of target genes.This study can lay the foundation for the further study of the molecular regulation mechanism of heat shock proteins in diapause insects.