苦碟子中对香豆酰转移酶基因克隆分析与功能验证  

Genetic Cloning and Functional Verification of Hydroxycinnamoyltransferase in Ixeris sonchifolia Hance

作　　者：李天真 周威[2] 殷华[2] 张同存[1] 刘涛[2] LI Tian-zhen;ZHOU Wei;YIN Hua;ZHANG Tong-cun;LIU Tao(School of Biological Engineering,Tianjin University of Science &Technology,Tianjin300457;Tianjin Institute oflndustrial Biotechnology,Chinese AcademyofSeiences,Tianjin 300308)

机构地区：[1]天津科技大学,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308

出　　处：《生物技术通报》2018年第11期120-126,共7页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(31770104)

摘　　要：苦碟子含有绿原酸等多种酚酸类化合物,该类化合物具有抗氧化和清除自由基等生物活性。对香豆酰转移酶(Hydroxycinnamoyltransferase,HCT)是此类化合物生物合成途径中的关键酶。本研究以苦碟子叶片为实验材料,采用转录组测序、序列分析以及RT-PCR的方法,获得一条对香豆酰转移酶基因,命名为IsHCT(GenBanK:MH151086)。序列分析表明该基因长1 320 bp,编码439个氨基酸。通过氨基酸序列比对及生物信息学分析发现,IsHCT氨基酸序列含有两个保守结构域,属于BAHD家族中的HCT酶类。系统发育进化树结果表明,该序列与菊苣的CiHCT的同源性最高,序列相似性高达95%。通过在大肠杆菌中过表达IsHCT和来源于拟南芥的对香豆酰辅酶A连接酶(At4CL),并在发酵培养基中饲喂底物咖啡酸和奎尼酸,在发酵液中检测到绿原酸的合成,表明IsHCT能够催化咖啡酰辅酶A与奎尼酸合成绿原酸,具有对香豆酰转移酶功能。Phenolic acid compounds from Ixeris sonchifolia Hance,such as chlorogenic acid,etc.,are reported to have antioxidationand free radicals scavenging activities. Hydroxycinnamoyltransferase(HCT)is a key enzyme in the biosynthetic pathway of plant chlorogenicacid. A hydroxycinnamoyltransferase gene from I. sonchifolia Hance leaves was cloned using transcriptome sequencing,sequence analysisand RT-PCR strategies,and named as IsHCT(GenBank accession number :MH151086). The open read frame of the gene was 1320 bpencoding a putative protein of 439 amino acids. Multiple sequence alignment and bioinformatics analysis results showed that IsHCT containedtwo conserved domains,and belonged to the HCT enzyme in the BAHD family. Phylogenetic tree analysis demonstrated that this gene had thehighest homology with CiHCT from Cichorium intybus,and the similarity reached 95%.Finally,by overexpressing IsHCT and At4 CL fromArabidopsis thaliana in Escherichia coli and feeding the substrates caffeic acid and quinic acid in the fermentation medium,and chlorogenicacid was detected in the fermentation liquid,indicating that the IsHCT catalyzed the chlorogenic acid synthesis from caffeyl coenzyme A andquinic acid,i.e.,it functioned as HCT

关 键 词：苦碟子 对香豆酰转移酶 绿原酸 大肠杆菌 

分 类 号：Q943.2[生物学—植物学] S567.239[农业科学—中草药栽培]