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.
出处
《生物技术通报》
CAS
CSCD
北大核心
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)
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
Myrmecia incisa Arachidonic acid Fatty acid elongase Agrobacterium tumefaciens Arabidopsis thaliana
