水稻线粒体26S rRNA基因在大肠杆菌JM101中的克隆及分子鉴定

用BT型水稻喜峰A黄化苗为材料,按本实验过去报道经修改后的方法提取线粒体DNA,经EcoR1完全酶切后,随机克隆到pUC19载体上,转化大肠杆菌,在含氨苄青霉素(50μg/m1)和X-gal的LB固体平板上筛选白色转化子。随机提取重组子DNA,以玉米26S rRNA基因为探针,经Southern分子杂交鉴定,一个插入1.3kb水稻线粒体DNA片段的重组质粒杂交结果为阳性,并将这个含有26S rRNA基因片段的重组质粒命名为pXMT1。

MOLECULAR CLONING AND DETERMINATION OF RICE MITOCHONDRIA 26s rRNA GENE IN ESCHERICHIA COLI JM101

Mitochondria DNA was isolated from darkgrown shoots of Xi Feng A according to the procedure reported previously. Mitochondria DNA was digested with EcoR1 completely, and ligated with pUC19 vector DNA which was digested with EcoR1. The ligated DNA was transformed into E. coli JM101. The transformants were selected on LB medium which containing ampicillin 50ug/ml. All of the bacterial clones were hybridized with 32P-labelled maize 26S rRNA gene, seven positive clones were obtained. The plasmid DNAs were isolated from the clones and digested with EcoR1 and southern transferred to NC paper. The membrance was hybridized with maize 26S rRNA probe. One clone which a shortest hybridization band (1.3kb) was named pXMT1.