麻疯树JcFATA基因干涉表达载体构建和功能初步分析  

Construction of Interference Expression Vector and Primary Function Analysis of JcFATA Gene in Jatropha curcas

作　　者：刘颖[1] 刘国选[1] 黄川腾[2] 杨亚利[3] 唐家年 何春英 林诗婉 高美芳[1] 吴东霖[1] 陈建平[4] LIU Ying;LIU Guo-Xuan;HUANG Chuan- Teng;YANG Ya- Li;TANG Jia-Nian;HE Chun-Ying;LIN Shi-Wan;GAO Mei-Fang;WU Dong-Lin;CHEN Jian-Ping(Faculty of Agricultural Science, Guangdong Ocean University, Zhanjiang 524088, China;Hainan Provincial Forestry Institute, Haikou 571000, China;School of Medicine, Jiaying College, Meizhou 514015, China;College of Food and Technology, Guangdong Ocean University, Zhanjiang 524088, China)

机构地区：[1]广东海洋大学农学院,湛江524088 [2]海南省林业科学研究所,海口571000 [3]嘉应学院医学院,梅州514015 [4]广东海洋大学食品科技学院,湛江524088

出　　处：《农业生物技术学报》2018年第5期899-910,共12页Journal of Agricultural Biotechnology

基　　金：广东省教育厅创新强校项目(No.2016KQNCX067);湛江市科技攻关项目(No.2016B101);广东海洋大学博士科研启动项目(No.R17023);国家级大学生创新创业训练计划项目(No.CXXL2016015);省级大学生创新创业训练计划项目(No.CXXL2017045)

摘　　要：植物脂酰-酰基载体蛋白硫酯酶A(fatty acyl-acyl carrier protein thioesterase,FATA)是脂肪酸积累过程中的关键酶,直接调控脂肪酸的含量与组成。目前有关麻疯树(Jatropha curcas)FATA基因(JcFATA)(Gen Bank登录号:EU267122.2)的功能研究还未见报道。本研究首先构建JcFATA基因RNA干涉(RNA interference,RNAi)表达载体,进行下调表达研究,以麻疯树叶片cDNA为模板,克隆出JcFATA基因正向片段FATA1(439 bp),然后将其连接到含内含子的干涉表达载体p YLRNAi.2-35S上,构建JcFATA基因RNAi表达载体pYLRNAi.2-35S-FATA1-FATA2;再通过电击法将其转入到根癌农杆菌(Agrobacterium tumefaciens)EHA105感受态细胞中,然后利用花序侵染法进行拟南芥(Arabidopsis thaliana)遗传转化,最后通过PCR检测和Southern blot验证。对转基因拟南芥植株进行了表型观察、表达量分析和脂肪酸的含量测定,结果显示,JcFATA基因干涉表达的转基因植物生长发育可能受到抑制。本研究结果可为进一步探讨JcFATA基因的功能及该基因在麻疯树遗传改良中的应用提供理论依据。The fatty acyl-acyl carrier protein thioesterase （FATA） is a key enzyme in the accumulation of fatty acids, which directly regulates the content and composition of fatty acids. The function of the acyl-ACP thioesterase A gene （GenBank accession number： EU267122.2） in Jatropha curcas has not been reported before. In this study, the JcFATA gene RNAi expression vector had been constructed and down-regulation of JcFATA gene expression was accomplished, the positive fragment FATA1 （439 bp） of JcFATA gene was cloned from Jatropha curcas leaf cDNA , which was then inserted into pYL RNAi.2-35S contained intron to construct the intermediate vector pYLRNAi.2-35S-FATA1; the reverse fragment FATA2 （439 bp） was amplified by PCR used the intermediate vector as a template, and then inserted into the intermediate vector to construct the JcFATA gene RNAi expression vector pYLRNAi.2-35S-FATA1-FATA2; it was transformed into Agrobacterium tumefaciens EHA105 competent cells by electroporation, and then transformed into Arabidopsis thaliana by inflorescence infection method. Finally, the transformation was confirmed by PCR and Southern blot. In addition, preliminary phenotypic observation, expression analysis and determination of fatty acid content of transgenic Arabidopsis plants was carried out in this study, the results indicated that the growth and development of transgenic plants might be inhibited by RNAi expression of JcFATA gene. The results of this study can provide a theoretical basis for further discussion of the function of JcFATA gene and its application in the genetic improvement of Jatropha curcas.

关 键 词：麻疯树  麻疯树脂酰-酰基载体蛋白硫酯酶A基因(JcFATA)  RNA干涉(RNAi)  拟南芥转化  脂肪酸 

分 类 号：S72[农业科学—林木遗传育种] Q75[农业科学—林学]