叶用莴苣热激蛋白90(LsHsp90)基因的克隆及其在热激下的表达

Molecular Cloning and Expression Analysis of Heat-Shock-Protein90 (LsHsp90) Gene from Leaf Lettuce (Lactuca sativa L.) Under Heat Shock

【目的】克隆叶用莴苣热激蛋白Hsp90基因并探讨其响应热胁迫的相关机制,为揭示叶用莴苣抗热分子机制奠定理论基础。【方法】采用同源克隆和RACE技术相结合,从叶用莴苣叶片总RNA中克隆LsHsp90 cDNA序列。通过实时荧光定量PCR(qRT-PCR)分析LsHsp90在叶用莴苣耐热品种Z36和热敏品种S106受37℃和42℃热激后叶片的表达差异。【结果】该基因cDNA序列全长2 330 bp,开放阅读框2 097 bp,编码698个氨基酸,推导的蛋白质分子量约79.8 kD。蛋白质结构预测及同源比对分析表明,LsHsp90基因编码蛋白含ATPase位点和Hsp90保守结构域,并与拟南芥(AY081302.1)、紫茎泽兰(EU269070.1)等多种物种的热激蛋白90高度同源;进化树分析表明,LsHsp90与紫茎泽兰Hsp90基因(EU269070.1)聚为一类。qRT-PCR分析表明,37℃热胁迫下,该基因在耐热品种和热敏品种的叶片中均上调表达;42℃高温胁迫下,热敏品种S106中下调表达,而耐热品种Z36上调表达。【结论】成功从叶用莴苣叶片中分离克隆到LsHsp90,该基因具有已知物种Hsp90基因的特征,该基因在热胁迫下有不同的表达特征,预示该基因其可能在抗高温胁迫中起重要作用。

【Objective】 LsHsp90 gene was cloned and used to analyze the expression profiles under heat shock stresses,in order to provide a foundation for investigating heat tolerant mechanisms of molecular regulation in stress tolerance of Lactuca sativa L.【Method】 With homology cloning approaches coupling with RACE techniques,the full-length cDNA of LsHsp90 was cloned from total RNA of the leaves of L.sativa L.Real-time quantitative PCR（qRT-PCR） was used to analyze the expression patterns of this gene in leaves after heat shock unter 37℃ and 42℃.【Result】The full cDNA sequence of LsHsp90 is 2 330 bp,containing a 2 097 bp open reading flame（ORF） and encoding 698 amino acid residues with a predicted molecular mass of 79.8 kD.The LsHsp90 protein contains a predicted ATPase site and a Hsp90 conservative structure domain,which is highly conserved in plants and especially similar to Hsp90 from Ageratina adenophora（EU269070.1） and Arabidopsis thaliana（AY081302.1）.Phylogenetic analysis showed that LsHsp90 and Hsp90 from Ageratina adenophora（EU269070.1） were clustered into one group.Real-time PCR analysis showed that,the expression of LsHsp90 in leaves of two varieties was upregulated under 37℃.LsHsp90 was induced down regulation in heat-sensitive variety S106 under 42℃,while the expression of LsHsp90 in heat-tolerant variety Z36 was upregulated.【Conclusion】LsHsp90 gene was firstly isolated and characterized from L.sativa L.This gene has genetic characteristics similar with all species and the induced expression profiling of LsHsp90 showed different patterns under heat stress.The results indicate that the LsHsp90 gene may play an important role in response to heat shock stress.