摘要
甘氨酸甜菜碱是植物体内重要的渗透调节物质,通过逐步克隆方法,将甜菜碱合成途径中编码三个关键酶(PEAMT、CMO和BADH)的基因构建到同一表达载体中,并经过酶切检测,证明得到了正确表达载体质粒———pCBP。将pCBP转入农杆菌GV3101,用真空渗透法转化拟南芥(Col 0)植株,通过抗性筛选获得了转基因植株,PCR检测证实三个目的基因已经转入到拟南芥植株中。抗盐性检测表明,在含NaCl125mmol·L-1的1/2MS培养基上,转基因植株的生长明显优于Col 0。
Glycine betaine (GlyBet) is an established target for metabolic engineering of stress tolerance because it is a potent osmoprotectant that many plants lack. GlyBet is synthesized in the chloroplast by a two-step oxidation of choline (Cho) that is catalyzed by CMO and BADH. Cho is one of essential precursors for the synthesis of GlyBet. PEAMT is a key enzyme in the Cho biosynthetic pathway and serves as a step-limiting controller. The CMO cDNA and BADH cDNA drived by 35S and the PEAMT genomic DNA were cloned into the binary vector(pCAMBIA1300) containing HPTⅡgene step by step. The plasmid was checked by 7 enzymes, and the fingerprints were expected. The plasmid of this expressing vector was transferred into the Agrobacterium tumefaciens (GV3101) by electroporation, then used in genetic transformation of A. thaliana mediated by vacuum infiltration. The primary transgenic plants were selected for hygemycin resistance and verified by PCR. The transgenic lines showed less growth inhibitation than control on 1/2MS containing 125 mmol·L^(-1) NaCl.
出处
《林业科学研究》
CSCD
北大核心
2005年第3期231-235,共5页
Forest Research
基金
科技部转基因专项:"抗干旱耐盐碱基因工程麻黄新品种培育(J2002 B 007)"
科技部973项目:"林木育种的分子机理"(G199916003)
关键词
甜菜碱
表达载体
抗盐
GlyBet
expressing vector
salt resistance
