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沙葱萤叶甲热激蛋白基因GdHsp10a的克隆、分子特征与表达分析

引用本文：陈龙,谭瑶,周晓榕,庞保平,单艳敏,张卓然.沙葱萤叶甲热激蛋白基因GdHsp10a的克隆、分子特征与表达分析.植物保护学报,2019,46(2):417-424

DOI：10.13802/j.cnki.zwbhxb.2019.2017218

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作者	单位	E-mail
陈龙	内蒙古农业大学, 草原昆虫研究中心, 呼和浩特 010019
谭瑶	内蒙古农业大学, 草原昆虫研究中心, 呼和浩特 010019
周晓榕	内蒙古农业大学, 草原昆虫研究中心, 呼和浩特 010019
庞保平	内蒙古农业大学, 草原昆虫研究中心, 呼和浩特 010019	pangbp@imau.edu.cn
单艳敏	内蒙古自治区草原工作站, 呼和浩特 010020
张卓然	内蒙古自治区草原工作站, 呼和浩特 010020

中文摘要:为明确热激蛋白HSP10在沙葱萤叶甲Galeruca daurica生长发育过程中的作用，采用RTPCR及RACE技术克隆沙葱萤叶甲Hsp10基因cDNA的全长序列，采用生物信息学方法分析其序列特征，并利用qPCR技术对该基因在沙葱萤叶甲不同发育阶段、成虫羽化后不同时期以及不同温度下的表达谱进行分析。结果表明，克隆获得1条新的沙葱萤叶甲Hsp10基因，命名为GdHsp10a，GenBank登录号为MG460308，cDNA序列全长为526 bp，开放阅读框为333 bp，编码蛋白含110个氨基酸，预测分子量为11.97 kD，等电点为9.74；无信号肽及跨膜结构。同源比对及系统进化分析表明，GdHSP10a与光肩星天牛Anoplophora glabripennis HSP10亲缘关系最近，氨基酸序列一致性为53.15%。qPCR结果表明，GdHsp10a在沙葱萤叶甲各发育阶段均有表达，其中在卵期和成虫期的表达量显著高于幼虫期、预蛹期及蛹期；成虫羽化后不同时期表达量差异显著，25 d时表达量最高，其次为100、10和7 d时的表达量；温度对GdHsp10a表达量有显著影响，30℃下表达量最高，35℃下表达量次之，15、20、25及40℃下表达量最低且无显著差异。表明GdHsp10a可能在沙葱萤叶甲生长发育及成虫越夏中起着多重作用。

中文关键词:沙葱萤叶甲热激蛋白HSP10 基因克隆分子特性基因表达温度胁迫

Molecular cloning, characterization and expression analysis of the heat shock protein gene GdHsp10a in Galeruca daurica (Coleoptera: Chrysomelidae)

Author Name	Affiliation	E-mail
Chen Long	Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010019, Inner Mongolia Autonomous Region, China
Tan Yao	Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010019, Inner Mongolia Autonomous Region, China
Zhou Xiaorong	Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010019, Inner Mongolia Autonomous Region, China
Pang Baoping	Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010019, Inner Mongolia Autonomous Region, China	pangbp@imau.edu.cn
Shan Yanmin	Grassland Station of Inner Mongolia, Hohhot 010020, Inner Mongolia Autonomous Region, China
Zhang Zhuoran	Grassland Station of Inner Mongolia, Hohhot 010020, Inner Mongolia Autonomous Region, China

Abstract:In order to investigate the functions of HSP10 in the development of Galeruca daurica, a fulllength cDNA encoding HSP10 was cloned from G. daurica by RT-PCR and RACE technologies. Bioinformatics programs were applied to analyze its sequence characteristics. qPCR was used to detect its mRNA expression levels at various developmental stages of G. daurica, different periods after the adult eclosion, and different temperature stresses. The results showed that the complete cDNA of the gene encoding heat shock protein HSP10 was obtained from G. daurica, and named GdHsp10a (GenBank accession:MG460308). Its full-length cDNA was 526 bp containing a 333 bp open reading frame that encodes 110 amino acids with the predicted molecular weight of 11.97 kD and pI of 9.74. The deduced protein contained no signal peptide and transmembrane domain. Homology and phylogenetic analyses showed that the amino acid sequence of GdHSP10a had the highest similarity (53.15%) with that of Asian longhorn beetle Anoplophora glabripennis HSP10. GdHsp10a was expressed at all developmental stages of G. daurica, and the expression levels in eggs and adults were significantly higher than those in the larva, pre-pupa and pupa. The expression levels were significantly different among the different periods after the adult eclosion, and reached the highest on the 25th day. Temperature had a significant effect on the expression of GdHsp10a, with the maximum at 30℃, followed by at 35℃, and with the minimum at 15, 20, 25 and 40℃, among which no significant differences existed. The results suggested that GdHsp10a might play multiple roles in the development of G. daurica and its adult oversummering.

keywords:Galeruca daurica HSP10 gene cloning molecular characterization gene expression temperature stress

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