摘要
利用Ｍｕ转座子细胞内克隆了大肠杆菌海藻糖合成酶 ｏｔｓＢＡ基因，克隆频率为１．４５ ｘ １０(－３)／ Ｋａｎ(ｒ)转导子。经遗传互补、酶切和部分序列分析表明ｏｔｓＢＡ基因位于克隆质粒。亚克隆 ２．８７ｋｂ ＤＮＡ片段至不同拷贝数表达质粒并分别转化大肠杆菌ｏｔｓＢＡ基因缺失株，转化株恢复 在０．５ｍｏｌ／Ｌ ＮａＣｌ培养基上生长的功能，高渗透压诱导实验表明，转化株能够合成克隆基因 产物海藻糖，但合成量不受克隆质粒拷贝数影响。海藻糖良好的抗高渗能力可能在农作物育 种方面发挥重要作用。为构建含有海藻糖合成酶基因的植物表达载体，并在农杆菌的介导下 转入植物，赋予其抗高渗、耐干旱能力奠定了重要的研究基础。
Escherichia colt trehalose synthase otsBA genes have been cloned by using transposon Mu in vivo. The cloning frequency was l.45 X 10-3/ Kan r transductant Genetic complements, endonuclase digestion and partial nucleotide sequencing analysis of these clones indicated that otsBA gene was on plasmid Mud5005. Subcloning 2.87kb DNA fragment onto expression plasmids with higher or lower copy number and transforming into E. coli recipient strain FF4050, which have otsBA genes deleted respectively, the transformed strains obtained ability of growth on medium containing 0.5mol/ L NaCl and increased trehalose synthesis under high osmoic pressure. Production and accumulation of trehalose may play an important role in crop breeding. These studies will lay an important foundation for constructing an expression vector with the otsBA genes and transformation to tobacco mediated by Agrobacterium, and obtaining ability for drought resistance and high osmotic pressure tolerance.
基金
国家科委"九五"攻关项目!96-C03-01-09
