RFLP缩减杂交技术的改良与水稻雄性不育细胞质基因组差异片段的克隆

Isolation of polymorphic DNA fragments between normal and cytoplasmic male sterility lines of rice using a modified RFLP subtractive hybridization method

对基因组RFLP缩减杂交技术进行了改良,即用连接成大分子的DriverDNA与TesterDNA杂交,再利用PCR纯化试剂盒对大片段DNA回收率低的原理去除大部分DriverDNA以及与之杂交的非特异性TesterDNA片段.经过PCR扩增和克隆,获得多态性片段,可快速获得在亲本间有差异的分子标记.用该技术对水稻野败型雄性不育细胞质和正常细胞质DNA进行RFLP缩减杂交,获得了2个在不育系细胞质DNA与保持系细胞质DNA间的差异片段.

To obtain polymorphic markers between parents, a modified RFLP-subtractive hybridization method was developed. The method uses restriction fragments of a parent as a driver, which were ligated to form large fragment DNA, for hybridization with tester DNA, and the driver/tester hybrid fragments are removed by using a PCR purification kit. The method was used to isolate polymorphic sequences between Zhenshan97B and a Rf-gene near-isogenic line ZSR11 with normal and CMS-WA cytoplasms, respectively. As a result, two polymorphic sequences, Bgl II-1 and Pst I-4, were obtained. Southern blot analysis indicated that the polymorphisms were associated with the normal and CMS-WA types of cytoplasm.