马铃薯St4CL的克隆及表达分析

Cloning and Expression Analysis of St4CL Gene in Solanum tuberosum

4-香豆素辅酶A连接酶(4CL)是苯丙烷代谢途径中关键酶之一,克隆马铃薯4CL,并分析其表达情况,为马铃薯4CL的功能研究奠定基础。从马铃薯青薯9号中克隆St4CL,并进行生物信息学分析,采用qRT-PCR技术检测St4CL在青薯9号各组织中以及不同马铃薯品种中的表达量。结果表明,St4CL的CDS长度为1710 bp,编码569个氨基酸,蛋白分子质量约为61.83 kD,等电点为5.53。生物信息学分析表明,St4CL蛋白是稳定的疏水性蛋白,具有无规则卷曲(42.36%)、α-螺旋(28.47%)、延伸连(21%)、β-折叠(8.08%);亚细胞定位预测显示,St4CL位于叶绿体类囊体膜上;系统进化分析表明,St4CL蛋白与番茄的Sl4CL蛋白亲缘关系最近,二者同源性为97%。St4CL启动子区域含有多种与逆境响应相关的顺式作用元件,如脱落酸响应元件、赤霉素响应元件以及与干旱和黄酮类生物合成相关的MYB结合位点。qRT-PCR结果显示,St4CL在青薯9号各组织中均有表达,叶片中表达量最高,花中次之,根中表达量最低;在不同马铃薯品种中的表达量存在差异,在高抗晚疫病品种青薯10号中表达量最高,感病品种下寨65中的表达量最低。

4-coumarate:CoA ligase(4 CL)is one of the most important enzymes in phenylpropane metabolism,thus cloning 4 CL from Solanum tuberosum and analyzing its expression will lay foundation for studying its function.A gene named St4 CL was cloned from Qingshu 9,bioinformatics for it was conducted,and qRT-PCR was used to detect the expressions of St4 CL in the different tissues and varieties.The results demonstrated that the CDS of St4 CL gene was 1710 bp,and it encoded 569 amino acids.The molecular weight of protein was 61.83 kD,and the isoelectric point was 5.53.Bioinformatics analysis showed that St4 CL was a stable hydrophobic protein with random coil(42.36%),α-helix(28.47%),extended strand(21%),andβ-turn(8.08%).Subcellular localization prediction showed that St4 CL was located on the thylakoid membrane of chloroplast.Phylogenetic analysis revealed that St4 CL protein had the highest sequence similarity(about 97%)with and the closest genetic relationship to Sl4 CL in Solanum lycopersicum.The promoter of St4 CL gene contained many cis-acting elements related to stress response,such as abscisic acid responsive element,gibberellin-responsive element,MYB binding site involved in drought and flavonoid biosynthesis,etc.qRT-PCR analysis showed that St4 CL expressed in all tissues,the highest in leaves followed by flowers,and the lowest in roots.The expression of St4 CL varied in different varieties,with the highest expression in Qingshu10 and the lowest expression level in Xiazhai 65.