摘要
实验选用油菜湘矮不育系及同核保持系叶绿体DNA为材料,用4种限制性内切酶EcoRⅠ、BamHⅠ、PstⅠ和SmaⅠ完全酶解。在EcoRⅠ酶解的保持系ctDNA图式中,观察到唯一的差异片段E3.2kb。将此片段连接于载体pUC9进行克隆,经克隆杂交及电泳分析鉴定,获得重组子。以rRNA基因为探针,与EcoRⅠ酶解的保持系ctDNA杂交,其中一条阳性带显示在差异片段E3.2。进一步以E3.2片段为探针,与经过限制酶BamHⅠ、EcoRⅠ、salⅠ、BgⅢ、HindⅢ和PstⅠ酶解后的rRNA基因反杂交。根据rRNA基因图谱,这一与不育性有关的E3.2kb片段被定位于距16S rRNA基因5'端+2.0至+5.4kb的前导序列中。此结果表明,这段可能与花粉育性形成有关的片段定位于叶绿体基因组反向重复区。
The ctDNA of sterile line and its maintainer from Brassica napus L. var. xiangai were digested by restriction endonucleases EcoRI, BamHl, Pstl and Smal. Only one special fragment E3.2kb was observed in EcoRI restriction pattern of maintainer. After being eluted, this fragment was incubated with plasmid pUC9 and then transformed E. coli JM83. Through colour screening, clony hybridization and electrophoretic analyses, the special clone carrying E3.2 was obtained. The rRNA gene probe was used to hybridize with the EcoRl restriction pattern. The result showed that E3.2kb fragment is homologous to rRNA gene. And then, we use E3.2 fragment as probe to hybridize with rRNA gene digested by BamHI, EcoRI. SalⅡ, BglⅡ, HindⅢ and Pal. According to rRNA gene map, E3.2 fragment was located at the leader sequence of 16S rRNA gene, from + 2.0kb to + 5.4kb. Because this 3.4kb region was in the inverted repeat region of ctDNA and it is homologous to E3.2kb that related to CMS. So probably CMS is related to this 3.4kb region in the inverted repeat region of ctDNA.
