RolC基因的克隆及细胞分裂素在烟草中的过量表达

THE CLONING OF ROLC GENE AND OVER EXPRESSION OF CYTOKININS IN NICOTIANA TABACUM

用ＰＣＲ方法从发根农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ）１６０１质粒中扩增出ｒｏｌＣ基因，并构建ＣａＭＶ３５Ｓ启动子驱动下的ｒｏｌＣ基因表达质粒ｐＣａＲ。以农杆菌介导的叶盘法，分别对野生型烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．ｃｖ．Ｗ３８）和已经异戊烯基转移酶基因（ｉｐｔ）转化的烟草进行了转化。Ｓｏｕｔｈｅｒｎｂｌｏｔ和ＲＮＡＤｏｔｂｌｏｔ分析表明，ｒｏｌＣ基因已转入烟草植株，并且可以正常表达。观察发现，经ｐＣａＲ转化的烟草，其形态特征与细胞分裂素过量表达时植株表现出的特征一致。用ＥＬＩＳＡ方法测定转基因烟草植株中激素含量，结果显示，单独转ｒｏｌＣ基因烟草和转ｒｏｌＣ和ｉｐｔ两个基因的烟草，细胞分裂素水平有不同程度的提高。转基因烟草表现多芽、节间距缩短等特征。

Using PCR method the rolC gene was amplified from Agrobacterium rhizogenes,and CaMV 35S/rolC expression vector pCaR was constructed. The chimeric gene via agrobacterium mediated procedure was transformed separately into the wild type tobacco (Nicotiana tabacum L. cv. W38) and the transgenic tobacco of ipt gene. The putative transgenic plants were assayed with Southern blot and RNA Dot blot analysis. The observation suggested that the transgenic tobacco exhibited the abnormal phenotypes as a consequence of the overproduction of cytokinins. Whereas the ELISA assay indicated that the cytokinins level increased separately in transgenic plants. The growth of the transgenic plants show multiple budding of shoots with short internodal length.