叶用莴苣LsTMK1基因的克隆及高温胁迫条件下的表达分析

Cloning and expression analysis of LsTMK1 gene in lettuce(Lactuca sative L.) under high temperature stress

【目的】为探明高温诱导叶用莴苣过早抽薹的原因。【方法】克隆测序得到LsTMK1的CDS全长序列,并利用ExPaSy,InterProScan,SignalP 4.1Server,TMpred Server,MEGA7.0等软件及在线工具对该基因进行生物信息学分析,使用qRT-PCR技术对该基因的表达量进行测定。【结果】生物信息学分析表明,LsTMK1开放阅读框为2 829bp,编码942个氨基酸。分子量约为103kD,理论等电点(pI)5.78。蛋白结构分析表明其含有14个保守结构域,3个跨膜螺旋结构,在26～27之间具有信号肽切割位点,通过D值判断该蛋白为分泌蛋白。DNAMAN同源性分析表明,LsTMK1氨基酸序列与朝鲜蓟CcTMK1 (登录号:KVI08578.1)和向日葵HaTMK1 (登录号:XM_022181262.1)的亲缘关系较近,相似性较高。实时荧光定量PCR结果显示,经高温处理后8、16、24、32d和40d的叶用莴苣茎内LsTMK1的表达量均出现上调。【结论】LsTMK1基因可能与高温诱导叶用莴苣提前抽薹密切相关。

【Objective】 In order to explore the factors which lead to premature bolting of leaf lettuce at high temperature, the gene of LsTMK1 which selected from differently expressed proteins was cloned and analyzed. 【Method】 The full-length CDS of LsTMK1 gene was obtained by gene cloning. The bioinformatics analysis of the gene was carried out by software such as ExPaSy, InterProScan, SignalP 4.1 Server, TMpred Server, MEGA7.0 and online tools. The expression level was analyzed by real-time quantitative PCR..【Result】 Bioinformatics analysis showed that the LsTMK1 open reading frame was 2 829 bp and encoding 942 amino acids. The molecular weight was about 103 kD and the theoretical isoelectric point （pI） was 5.78. The protein structure analysis showed that it contained 14 conserved domains with 3 transmembrane helix structures, with a signal peptide cleavage site between 26 and 27, and the protein was judged as a secreted protein by the D value. DNAMAN homology analysis showed that the amino acid sequence of LsTMK1 was relatively close to the artichoke CcTMK1 （accession number： KVI08578.1） and the sunflower HaTMK1 （accession number： XM_022181262.1）, and the similarity is high. Real-time PCR results showed that LsTMK1 expression was up-regulated in lettuce stems treated by high temperature on 8 days, 16 days, 24 days, 32 days and 40 days. 【Conclusion】 The LsTMK1 gene may be closely related to the bolting of leaf lettuce under high temperature stress.