雷公藤鲨烯环氧酶基因克隆与表达分析

摘要

Two squalene epoxidase (SE) genes were cloned by reverse transcription polymerase chain reaction (RT-PCR) from Tripterygium wilfordii hairy root.The genes are named as TwSE1 (GenBank accession MG717395) and TwSE2 (GenBank accession MG717396).Sequence analysis indicates that the open reading frame of TwSE1 and TwSE2 genes were 1 578 bp and 1 584 bp,which encoding 525 and 527 amino acids.Two amino acid sequences from TwSE1 and TwSE2 shared 76.18％ similarity,but were highly divergent in N-terminal regions.Further,gene expression pattern in different tissues and the expression level of TwSE in T.wilfordii hairy root after treatment by methyl jasmonate (MeJA) was detected by real-time PCR.TwSE1 and TwSE2 genes were expressed in roots,stems,young leaves,old leaves and flowers of T.wilfordii.TwSE1 expressed the highest in flowers and lowest in roots,but TwSE2 expressed the highest in flowers and lowest in old leaves.The expression of TwSE2 was lower than that of TwSE1 in all tissues.MeJA treatment enhanced the TwSE1 and TwSE2 expression in hairy roots.The expression level of TwSE1 and TwSE2 has a increasing tendency in the early stage,then decreased in the middle stage and increased in the late time.The highest expression was reached after the treatment of 3 h,then decreased,and the expression level increased rapidly after 12 h.In same treatment condition,the increase of TwSE2 gene expression level was less than that of TwSE1 gene.%以雷公藤(Tripterygium wil fordii Hook.f.)发状根为试验材料,采用RT-PCR方法,克隆得到2个雷公藤鲨烯环氧酶编码基因,命名为TwSE1(GenBank登录号MG717395)和TwSE2(GenBank登录号MG717396).序列分析表明,TwSE1和TwSE2的开放阅读框分别为1 578和1 584 bp,分别编码525和527个氨基酸,2条序列相似性为76.18％,但N端序列不保守.实时荧光定量PCR检测雷公藤鲨烯环氧酶在不同组织部位的表达模式,以及雷公藤发状根受茉莉酸甲酯(MeJA)诱导后基因表达的结果表明,TwSE1、TwSE2基因在雷公藤根、茎、嫩叶、老叶、花中均有表达,TwSE1在花中表达丰度最高,在根中表达丰度最低;但TwSE2在花和嫩叶中表达量最高,在老叶中表达量最低,且TwSE2在各组织部位的表达量都低于TwSE1.雷公藤发状根经MeJA诱导后,TwSE1、TwSE2基因表达量均上升,都表现为表达量先上升后下降再上升的趋势,并于诱导后3h达到一个高水平表达,之后下降,在诱导12 h后表达量又迅速上升;在相同诱导条件下,TwSE2基因表达水平的提高小于TwSE1基因.