胡椒4-香豆酸:辅酶A连接酶Pn4CL基因的克隆及表达分析  

Cloning and Expression Analysis of 4-coumarate:coenzyme A Ligase Gene(Pn4CL)in Piper nigrum

作　　者：范睿[1,2] 胡丽松 伍宝朵[1,3] 郝朝运 FAN Rui;HU Lisong;WU Baoduo;HAO Chaoyun(Spice and Beverage Research Institute,Chinese Academy of Tropical Agricultural Sciences,Wanning,Hainan 571533,China;Key Laboratory of Genetic Resources Utilization of Spice and Beverage Crops,Ministry of Agriculture and Rural Affairs,Wanning,Hainan 571533,China;Hainan Provincial Key Laboratory of Genetic Improvement and Quality Regulation for Tropical Spice and Beverage Crops,Wanning,Hainan 571533,China)

机构地区：[1]中国热带农业科学院香料饮料研究所,海南万宁571533 [2]农业农村部香辛饮料作物遗传资源利用重点实验室,海南万宁571533 [3]海南省热带香辛饮料作物遗传改良与品质调控重点实验室,海南万宁571533

出　　处：《热带作物学报》2020年第4期737-744,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金青年科学基金项目(No.31601626);中国热带农业科学院基本科研业务费(No.1630142017010)。

摘　　要：根据胡椒4-香豆酸:辅酶A连接酶(4-coumarate:coenzyme A ligase,4CL)基因的部分序列设计引物,运用RACE方法获得其家族成员的1个全长c DNA,命名为Pn4cl,长度2130 bp,开放阅读框1638 bp,编码545个氨基酸。预测Pn4CL分子量为59.57 k Da,理论等电点为5.70。该基因含有AMP-binding(AMP-binding enzyme)、Cai C[Acyl-Co A synthetase(AMP-forming)/AMP-acid ligaseⅡ]、PLN02246、AFD-classⅠ等结合域,具有植物4CL所共有的保守结构域。系统进化分析表明,Pn4CL与北细辛的同源性最高,同时与木兰分支类植物的4CL聚类在一起,与菊分支的进化距离较近,与蔷薇分支的进化距离较远。亚细胞定位表明,该蛋白定位在细胞膜上。Real-timeRT-PCR结果表明,该基因受外援激素SA和MeJA诱导表达,同时接种辣椒疫霉菌后,Pn4CL基因的表达量在抗/感2种胡椒中均出现先增加后减少的现象,并且在抗病种质中表达量较高。研究结果为Pn4CL的功能研究提供了理论依据。The full-length cDNA encoding 4-coumarate:coenzyme A ligase(4CL),designated as Pn4CL,was isolated from black pepper using RACE.The sequence of Pn4CL was 2130 bp in length,containing a 1638 bp open reading frame,and encoding a polypeptide of 545 amino acids with a calculated molecular weight of 59.57 kDa and a PI of 5.70.The protein had four conserved domains of AMP-binding(AMP-binding enzyme),CaiC(Acyl-CoA synthetase(AMP-forming)/AMP-acid ligase),PLN02246,AFD-class Ⅰ.Pn4CL had a closer relationship with Asarum heterotropoides and far from the evolution of Rosids by phylogenetic analysis.The subcellular localization indicated that the protein was on the cell membrane.The expression of Pn4CL could be regulated by exogenous MeJA and SA.When infected with Phytophthora capsici Leon,the expression of Pn4CL was significantly higher in the resistant germplasm than that in the susceptible germplasm by real-time RT-PCR.

关 键 词：胡椒 4-香豆酸:辅酶A连接酶 克隆与表达 

分 类 号：S188[农业科学—农业基础科学]