香椿TsCAD1基因的克隆与非生物胁迫下的表达特性

Cloning and Expression Characteristics of TsCAD1 Gene under Abiotic Stress Condition in Toona sinensis

肉桂醇脱氢酶(CAD)是木质素生物合成的关键酶之一,为探讨肉桂醇脱氢酶基因在香椿抵抗非生物胁迫方面发挥的作用,本研究从香椿转录组数据中筛选并克隆得到1个CAD基因,其开放阅读框为1068 bp,共编码355个氨基酸,结构域分析发现香椿CAD蛋白含有1个NADP(H)辅酶结构域,2个Zn结合位点,系统进化树分析表明香椿CAD与柑橘CAD1亲缘关系最近,将其命名为TsCAD1。实时荧光定量PCR结果显示,TsCAD1在香椿幼苗根中的表达量显著高于茎与叶中的表达量;38℃高温胁迫24 h期间,TsCAD1在香椿叶片中的表达量呈现先降低后显著上升的趋势;4℃低温胁迫24 h期间,TsCAD1的表达呈现先上升后下降的趋势;200 g/L PEG6000干旱胁迫24 h期间,TsCAD1的表达趋势为先下降后上升又下降,但其表达水平均低于对照;200 mmol/L NaCl盐胁迫处理24 h期间,TsCAD1的表达表现为先下降后上升,其表达水平均低于对照。根据以上结果推测香椿TsCAD1基因可能在香椿的非生物胁迫防御机制方面发挥功能,该试验结果为通过分子生物技术改良香椿的栽培抗性提供了理论参考。

Cinnamol alcohol dehydrogenase(CAD)is one of the key enzymes in lignin biosynthesis.In order to explore the role of cinnamyl alcohol dehydrogenase gene in Toona sinensis resistance to abiotic stress,one CAD gene was cloned from Toona sinensis according to the transcription databases.The open reading frame of the gene was 1068 bp,and encoded a protein of 355 amino acids.Domain analysis showed the CAD protein of Toona sinensis contained 1 NADP(H)binding domain and 2 Zn binding sites,and phylogenetic tree analysis showed the CAD of Toona sinensis was most closely related to citrus CAD1,so the gene is named as TsCAD1.The qRT-PCR results showed TsCAD1 gene expression in roots was significantly higher than that in stems and leaves.The TsCAD1 gene expression decreased first and then increased significantly during 38℃heat treatment for 24 hours.The expression first increased and then decreased during 4℃cold treatment for 24 hours.The TsCAD1 gene expression decreased first,then increased,and then decreased again during 200 g/L PEG6000 drought stress treatment for 24 hours,but the relative expression level was lower than the control.The expression trend was firstly decreased and then increased but the relative expression level was also lower than the control during 200 mmol/L NaCl salt stress treatment for 24 hours.All these results revealed TsCAD1 gene might play an important role in abiotic defense mechanism.The study provided a theoretical reference for improving the cultivation resistance of Toona sinensis by molecular biotechnology.