缺刻缘绿藻脂肪酸延长酶基因在拟南芥中的表达分析  

Expression of a Fatty Acid Elongase Gene from Myrmecia incisa in Arabidopsis thaliana Mediated by Agrobacterium tumefaciens

作　　者：于水燕[1] 陈颢[2] 周志刚[1] 

机构地区：[1]上海海洋大学农业部水产种质资源与利用重点开放实验室上海高校水产养殖E-研究院,上海201306 [2]国家海洋局第一海洋研究所,青岛266061

出　　处：《生物技术通报》2011年第11期88-94,100,共8页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(30972243);国家高技术研究发展计划课题(2009AA064401);教育部留学回国人员科研启动基金资助项目;上海市教育委员会科研创新项目(09ZZ167);海洋生物学重点学科资助项目(J50701)

摘　　要：缺刻缘绿藻(Myrmecia incisa)系单细胞淡水绿藻,能够大量合成并积累花生四烯酸(arachidonic acid,ArA,20:4ω6),尤其在氮饥饿条件下。基于该绿藻中的延长酶基因序列构建双元表达载体,通过根癌农杆菌(Agrobacterium tumefaciens)介导侵染拟南芥(Arabidopisis thaliana),筛选得到携带MiFAE基因的拟南芥转化株。在转基因第3代(T3)植株中应用PCR扩增目的基因片段和GUS染色,分别在DNA、mRNA和表达水平上均成功地检测到MiFAE基因的存在。GC-MS对不同组织甲酯化的脂肪酸进行检测,结果表明,在转基因的拟南芥营养生长期的叶片中,十六碳三烯酸(hexadecaterienoic acid,C16:3^7,10,13)和α-亚麻酸(α-linolenic acid,C18:3^9,12,15,ALA)在总脂肪酸中的百分含量与对照组相比明显下降,分别由10.5%和41.5%降到1.8%和19.6%。结合GUS染色结果,推测这些减少的产物可能通过外源MiFAE基因的作用,直接参与了蜡质或角质的合成代谢途径。Myrmecia incisa is a green coccoid freshwater microalga,which is rich in arachidonic acid（ArA,C20： 4ω 6,Δ5,8,11,14）,especially grown under nitrogen starvation stress.Based on a cloned fatty acid elongase gene（MiFAE） from M.incisa,a binary vector,pC-MiFAE,was constructed from pCAMBIA1301.It was transformed into Arabidopsis thaliana mediated by Agrobacterium tumefaciens,and hygromycin-resistant plants were selected.The MiFAE gene was detected being integrated in the plant genomes of T3 generation transformants.It has been transcripted and translated normally as revealed by RT-PCR and GUS staining,respectively.GC-MS analysis of fatty acids of leaves in vegetative growth from transgenic plants showed that the hexadecaterienoic acid（C16：3^7,10,13） and α-linolenic acid（C18：3^9,12,15,ALA） decreased from 10.5% and 41.5% in wild type to 1.8% and 19.6%,respectively.Unfortunately,no corresponding elongated products of these two decreased fatty acids were detected by GC-MS.Regarding to the GUS staining result,these decreased fatty acids were supposed to participate in the other metabolic pathways such as the biosynthesis of wax or cutin via a step where MiFAE gene worked possibly.

关 键 词：缺刻缘绿藻 花生四烯酸 脂肪酸延长酶 农杆菌 拟南芥 

分 类 号：Q943.2[生物学—植物学]