大葱4-香豆酸辅酶A连接酶基因Af4CL的克隆及表达分析  

Cloning and Expression Analysis of 4-coumaric Acid Coenzyme A Ligase Gene Af4CL in Allium fistulosum

作　　者：李逸 徐欢欢 张宇辰 王永勤[2] 刘乐承[1] LI Yi;XU Huan-Huan;ZHANG Yu-Chen;WANG Yong-Qin;LIU Le-Cheng(School of horticulture,Yangtze University,Jingzhou 434025,China;Vegetable Research Center/Key Laboratory of Horticultural Crop Biology and Germplasm Creation in North China,Ministry of Agriculture and Rural Areas,Beijing Academy of Agriculture and Forestry,Beijing 100097,China)

机构地区：[1]长江大学园艺园林学院,荆州434025 [2]北京市农林科学院蔬菜研究中心/农业农村部华北地区园艺作物生物学与种质创制重点实验室,北京100097

出　　处：《农业生物技术学报》2023年第2期273-281,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972409);北京市农林科学院科技创新能力项目(KJCX20200113)。

摘　　要：4-香豆辅酶A连接酶(4-coumaric acid coenzyme A ligase, 4CL)是类黄酮合成途径的一个关键酶,催化羟基香豆酸形成香豆酰辅酶A,香豆酰辅酶A是植物类黄酮生物合成的起始底物。本研究以大葱(Allium fistulosum)为材料,克隆了编码大葱4-香豆酸辅酶A连接酶的基因Af4CL(GenBank NO.OP921007),并进行了生物信息学分析,同时对Af4CL基因进行亚细胞定位和原核表达系统分析;结果显示,Af4CL编码区全长1 644 bp,编码547个氨基酸;生物信息学分析表明,Af4CL具有2个保守结构域BoxⅠ和BoxⅡ;进化分析结果表明,大葱Af4CL与大蒜(Allium sativum) As4CL的亲缘关系最近。构建Pro35S:Af4CL-GFP载体,并利用根癌农杆菌(Agrobacterium tumefaciens)介导的烟草(Nicotiana tabacum)叶片瞬时表达系统对Af4CL的定位进行了分析表明,Af4CL定位于细胞膜;利用原核表达系统成功诱导出目的蛋白。本研究为大葱类黄酮代谢的深入研究及类黄酮体外合成提供了参考。4-coumaric acid coenzyme A ligase(4CL) is a key enzyme in the flavonoid synthesis pathway,catalyzing the hydroxycoumaric acid to the formation of coumaroyl-CoA, which is a key enzyme in plant flavonoid biosynthesis at the starting substrate. In this research, the welsh onion(Allium fistulosum) was used as the material to clone the Af4CL gene, which encoding the welsh onion 4-coumaric acid-CoA ligase, and the bioinformatics analysis were also carried out. At the same time, the localization and prokaryotic expression system of Af4CL gene were analyzed. The results showed that the full-length coding region of Af4CL was 1 644bp and it encoded 547 amino acids. The bioinformatics analysis showed that there were 2 conserved structural regions: Box Ⅰ and Box Ⅱ;Evolutionary analysis results indicated that Af4CL was most closely related to As4CL of garlic(Allium sativum). The 35S:Af4CL-GFP vector was constructed, and the localization of Af4CL was analyzed by Agrobacterium-mediated transient expression system in tobacco(Nicotiana tabacum) leaves.The results showed that Af4CL was localized in the cell membrane;and the target protein was successfully induced and purified by the prokaryotic expression system. In conclusion, Af4CL gene was successfully cloned and the bioinformation analysis were carried out. At the same time, the target protein was successfully induced. These studies provide reference for the study of flavonoid metabolism and in vitro synthesis of flavonoids in A. fistulosum.

关 键 词：大葱 4-香豆酸辅酶A连接酶(4CL) 生物信息学 亚细胞定位 表达分析 

分 类 号：S633[农业科学—蔬菜学]