叶用莴苣热激蛋白LsHsp70-1707基因的克隆及表达分析

Cloning and expression analysis of heat-shock-protein LsHsp70-1707 gene from leaf lettuce(Lactuca sativa L.)

为了解叶用莴苣热激蛋白LsHsp70-1707基因在高温下的作用机制,通过同源克隆及RACE技术克隆并获得叶用莴苣热激蛋白LsHsp70-1707基因全长cDNA序列,实时荧光定量PCR(qRT-PCR)分析基因在不同叶用莴苣品种中的表达量差异。该基因全长2 400bp(KP258179),开放阅读框为2 106bp,编码701个氨基酸,与西瓜(AAC03416.1)、牵牛花(ABZ04081.1)、黄瓜(CAA52149.1)、菠菜(AAB91471.1)等HSP70蛋白高度同源。实时荧光定量PCR结果表明:高温处理时,该基因在热敏品种中表达没有明显增加,随着处理时间的增加甚至表现为下调,而在耐热品种中,其表达上调。成功克隆得到叶用莴苣热激蛋白LsHsp70-1707基因,热胁迫下该基因在不同品种中的表达有一定的差异性,推测该基因可能与叶用莴苣耐热性相关。

This study aimed at finding out the heat-shock-protein LsHsp70-1707 gene＇s mechanism under high temperature in leaf lettuce（Lactuca sativa）.The full-length cDNA of LsHsp70-1707 was obtained by homologous cloning and RACE technology.Real-time quantitative PCR（qRT-PCR）was used to analyze the expression differences of Lactuca sativain different varieties.LsHsp70-1707 had a cDNA sequence with 2 400 bp in length containing an open reading frame of 2 106 bp,which encodes a deduced 701 amino acids.It was highly homologous with other varieties＇ HSP70 protein,such as Citrulluslanatus（AAC03416.1）,Ipomoea nil（ABZ04081.1）,Cucumissativus（CAA52149.1）and Spinaciaoleracea（AAB91471.1）.The Real-time PCR results showed that：Under high temperature,the expression of LsHsp70-1707 gene did not have obvious increase and even downregulated along with the increase of processing time in heat sensitive variety,while its expression level was upregulated in heat resistant variety.LsHsp70-1707 gene was cloned in this study,and its expression were different to a certain degree in different cultivars under heat stress.We assume that the gene may be associated with heat resistance of leaf lettuce.