红花脂肪酸脱氢酶基因(CtFAD2-1)启动子克隆及功能分析  

Cloning and Functional Analysis of Fatty Desaturase Gene(FAD2-1)Promoter from Safflower(Carthamus tinctorious)

作　　者：李丹丹 王庆[1] 许鑫 于景盛 吴卫[1] LI Dan-Dan;WANG Qing;XU Xin;YU Jing-Sheng;WU Wei(College of Agriculture,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Medicinal Plant Breeding and Planting in Guizhou,Guiyang 550025,China)

机构地区：[1]四川农业大学农学院,成都611130 [2]贵州省药用植物繁育与种植重点实验室,贵阳550025

出　　处：《农业生物技术学报》2021年第4期663-672,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(81274020);贵州省药用植物繁育与种植人才基地(黔人领发[2013]15号)。

摘　　要：亚油酸通过调节膜脂的流动性参与植物代谢和各种胁迫反应,是植物脂类中主要的多不饱和脂肪酸。脂肪酸脱氢酶(fatty acid desaturase 2,FAD2)是亚油酸生物合成的关键酶,由FAD2基因编码,红花(Carthamus tinctorius)种子CtFAD2-1基因是其亚油酸生物合成的主要基因。本研究采用染色体步移方法克隆了红花CtFAD2-1基因,构建了含不同缺失启动子片段(P1:821 bp;P2:564 bp;P3:230 bp;P4:60 bp)的植物表达载体,并稳定转化拟南芥(Arabidopsis thaliana),并利用转基因方法对其功能进行验证。结果发现,该启动子中存在热激响应元件(heat responsive element,HSE)、低温响应元件(low temperature response,LTR)、干旱响应元件(MYB response,MBS)等非生物胁迫应答相关顺式元件,以及损伤、真菌及茉莉酸甲酯等响应基序。通过构建CtFAD2-1基因启动子缺失表达载体并稳定转化拟南芥,转基因拟南芥的β-葡萄糖苷酸酶(glucuronidase,GUS)组织化学分析结果显示,GUS在拟南芥的花、成熟种子和角果壁均有表达,且对干旱、盐、冷、高温、伤口和脱落酸(abscisic acid,ABA)激素处理等均有响应,其中-230~-60 bp是拟南芥组织GUS活性的关键区域。红花CtFAD2-1基因启动子属于非种子特异型启动子,且受多种非生物胁迫诱导,这与其含有多种非生物胁迫响应元件相一致。本研究为CtFAD2-1基因启动子的利用和进一步研究CtFAD2-1基因的表达调控提供理论依据。Linoleic acid is main polyunsaturated fatty acids in plant lipids,which plays key roles in plant metabolism.Fatty desaturase 2(FAD2)is a key enzyme for linoleic acid biosynthesis,CtFAD2-1 gene has the highest activity and was mainly responsible for the biosynthesis of linoleic acid in safflower(Carthamus tinctorius)seed.In this study,an 821 bp promoter region of CtFAD2-1 gene from safflower was cloned.Four 5’-deletion constructs of 821 bp(P1),564 bp(P2),230 bp(P3),60 bp(P4)were constructed and functionally characterized in Arabidopsis thaliana by using transgenic approach.Computational analysis found that the abiotic stress responsive cis-elements like heat responsive element(HSE),low temperature response(LTR),MYB response(MBS)etc,as well as wound,fungus,methyljasmonate responsive motifs existed in the promoter.The histochemical staining of GUS revealed that the flower,mature seed and silique wall were stained.In addition,the transgenic plants treated by drought,salt,cold,high-temperature,wound and ABA hormone as well as control plants were also stained.The-230~-60 bp might be the key region for GUS activity in A.thaliana tissues.CtFAD2-1 gene was not seed-specific and it could be induced by an assortment of abiotic stresses.This research plays an indispensable role in furthering the research related to the regulation of CtFAD2-1 gene.

关 键 词：红花 脂肪酸脱氢酶 功能分析 启动子 

分 类 号：S567.9[农业科学—中草药栽培]