摘要
来源于细菌的新霉素磷酸转移酶基因(NPT),氯霉素乙酰转移酶基因(CAT)以及来源于昆虫的荧光素酶基因等都是用于研究根癌农杆菌转化植物的良好标记。我们利用氯霉素乙酰转移酶嵌合基因和来源于Ti质粒T-区DNA的tmr基因,构建了中间载体pBZ 6102,并通过植物基因工程载体pGV 3850,将氯霉素乙酰转移酶嵌合基因和tmr基因引入了植物细胞,并测到了表达,在抗氯霉素植物中测到了氯霉素乙酰转移酶活性。中间裁体pBZ 6102上还有Pst Ⅰ,Xba Ⅰ等单一的限制性内切酶位点,外源基因极易插入。转化植物F_1代的种子抗性分析表明,80%左右的种子都能在含氯霉素的培养基上正常萌发,它们的幼苗中都有氯霉素乙酰转移酶活性,证明CAT基因通过了减数分裂稳定地保留在植物细胞的基因组内。
Agrobacterium tumefaciens induces neop-lastic growths called crown gall tumours onmany dicotyledonous plants.Recently,it hasbeen reported that some monocotyledonousplants,especially some important cereal pla-nts,such as Zea mays,are also sensitive toit.There is no crown gall tumour production(only proliferation around the wound place)when the monocotyledonous plant inoculatedwith A.tumefaciens.So crown gall tunouris not the good marker for plant transforma-tion with Agrobacterium tumefaciens. Using gene 4 (tmr) of T-DNA and chi-merc CAT gene,we set up an intermediatevector pBZ 6102 and introduced these twogenes into tobacco by the plant gene vectorpGV 3850 which is a modified nopaline typeTi plasmid.Because of the function of gene4 which codes for cytokinin synthase,at theinfected place of tobacco stem produce shootswhich contain not only the genetic transfor-mation marker—nopaline,but also chloram-phenicol resistant.The chloramphenicol ace-tyltransferase activity has been detected intransformed tobacco plants. Analysis of F 1 generation of 10 trans-formed tobacco plants shows that about 80%are chloramphenical resistant in which chlo-ramphenicol acetyltransferase activity has alsobeen detected,These results suggest that thereis not only one T-DNA copy in the prima-rily transformed plant cell. In the intermediate vector pBZ6102,there are several unique restriction enzymesites such as PstI,XbaI,so it is easy to in-sert foreign gene into it.Using pBZ6102 asintermediate vector for plant cell transforma-tion,there are three markers for screening.The C 58 C 1 (pBZ 6111) is now widely usedin our laboratory for plant protoplast andtissue transformation.
出处
《实验生物学报》
CSCD
1989年第3期259-267,共9页
Acta Biologiae Experimentalis Sinica
关键词
转化烟草
中间载体
CAT基因
Intermediate vector.Chimeric CAT gene.Transformed tobacco.
