花楸树热激蛋白SpHSP70-2基因的克隆及表达分析  

Cloning and Expression Analysis of Heat Shock Protein SpHSP70-2 Gene in Sorbus pohuashanensis

作　　者：赵冬雪 刘聪聪 鲁仪增 常金财 郑健[1] ZHAO Dong-xue;LIU Cong-cong;LU Yi-zeng;CHANG Jin-cai;ZHENG Jian(Landscape and Architecture College,Beijing University of Agriculture,Beijing 102206,China;Shandong Provincial Center of Forest Tree Germplasm Resources,Jinan 250102,Shandong,China;Hulunbeier Institute of Forestry Science,Hulunbeier 021008,Inner Mongolia,China)

机构地区：[1]北京农学院园林学院,北京102206 [2]山东省林木种质中心,山东济南250102 [3]呼伦贝尔市林业科学研究所,内蒙古呼伦贝尔021008

出　　处：《西北林学院学报》2021年第5期42-50,共9页Journal of Northwest Forestry University

基　　金：国家自然科学基金(31770369);国家林木种质资源共享服务平台(2003DKA21003);城乡生态环境北京实验室建设项目(PXM2015_014207_000014)。

摘　　要：探究花楸树响应高温胁迫的分子机制,为遗传改良和耐热品种的选育提供基础,依托花楸树叶片转录组测序数据,采用反转录PCR(RT-PCR)方法克隆得到HSP70家族中的1个基因,命名为SpHSP70-2,同时对该基因进行了生物信息学分析,利用荧光定量PCR(qRT-PCR)方法对其组织特异性和应答高温胁迫的表达模式进行了分析。结果表明,经克隆得到的SpHSP70-2基因与花楸树叶片转录组测序数据的基因序列一致,SpHSP70-2开放阅读框(ORF)长度为1 704 bp,编码567个氨基酸。SpHSP70-2蛋白具有HSP70特征结构域:核苷酸结合结构域(NBD)和底物结合结构域(SBD);SpHSP70-2基因无内含子;SpHSP70-2二级结构中具有ɑ螺旋(32.63%)、延伸链(23.28%)、β转角(7.23%)和随机卷曲链(36.86%);SpHSP70-2蛋白为疏水性蛋白,有11个跨膜螺旋,无信号肽;SpHSP70-2与同科的苹果、白梨的HSP70同源性最高,为90%以上;SpHSP70-2在花楸树的果实中表达量最大,在茎、根中表达量次之,在花中的表达量最小;在42℃高温胁迫处理下,SpHSP70-2表达量呈现先升高后下降趋势,在2 h时达到极值,随后表达量略缓慢下降,说明SpHSP70-2基因参与调控花楸树对高温胁迫的响应。研究结果为今后花楸树响应热胁迫的分子机制研究和引种驯化方面提供基础。In order to further explore the molecular mechanism of Sorbus pohuashanensis leaf in response to high temperature stress and provide basis for future genetic improvement and breeding of heat-tolerant varieties, a gene in the HSP70 family, named SpHSP70-2,was cloned by RT-PCR based on the sequencing data of the S.pohuashanensis transcriptome in this study.The gene was bioinformatically analyzed and the expression patterns of tissue specificity and response to high temperature stress were analyzed by qRT-PCR method.The results showed that the ORF of SpHSP70-2 gene was 1 704 bp in length and encoded 567 amino acids.SpHSP70-2 protein had the characteristic domains of HSP70:NBD(nucleotide binding domain) and SBD(substrate binding domain).No introns were found in SpHSP70-2 gene.SpHSP70-2 protein presented the features of secondary structure: ɑ helix(32.63%),extended chain(23.28%),β rotation(7.23%) and random curly chain(36.86%).The protein was hydrophobic with 11 transmembrane helices and without signal peptide.It had the highest homology with HSP70 of apple and Chinese white pear in the same family(more than 90%).The highest expression level of SpHSP70-2 was in the fruit, and the least in flower.Under high temperature stress(42℃),the expression level of SpHSP70-2 showed increasing and then decreasing trends, with the maximum value at 2 h.These results would provide a basis for molecular mechanism study in response to heat stress and introduction of S.pohuashanensis in the future.

关 键 词：花楸树 热激蛋白 基因克隆 荧光定量PCR 

分 类 号：S792.25[农业科学—林木遗传育种]