ACS和ACO基因克隆及植物转化

ACS AND ACO GENE CLONING AND PLANT TRANSFORMATION

根据已知序列设计PCR引物，分别扩增氨基环丙烷羧酸合酶（ACS）和氨基环丙烷羧酸氧化酶（ACO）基因并克隆到中间载体，经限制性内切酶酶切图谱分析、部分序列分析以及Southern印迹鉴定后，将两个基因单个或相互串联后反向亚克隆至植物表达载体pBI121。经农杆菌LBA4404转化番茄子叶，在抗性培养基上得到8株ACS基因反义转化的生根小苗以ACS基因的酶切小片段作为探针，经Southern印迹分析，证明获得了两株阳性转化株。

According to the published sequences, PCR primers of lundnocyclopropane-1-lcarboxylate (ACS) and l-aminocyclopropane-lcarboxylate oxidase (ACO) gene of tomato amplification were designed. Fragments of ACS and ACO gene were generated. The products of PCR fragments and T-vector ligation were transformed into E. colt (DH5a).The recombinants were selected by blue-white colony screening and restriction analysis.After partial sequencing and Southern hybridization, the fragments and stringed products o f two genes were subcloned into plant gene expression vector pBllZI in antisense orientation. The antisense vectors were transformed into Agrobaterium tumefaciens LBA4404 by freeze-thaw method. Fourteendayxild cotyledon pieces were infected with Agrobacterium,and transformants were selected with 75 ig nil-l kanamycin. Southern blotting with 0.gkb fragments of ACS gene as probe provided that two positively transgenic plantletS were obtained.