黏类小麦细胞质雄性不育系mtDNA消减文库的构建

Construction of mtDNA Substractive Library in CMS Lines of Wheat with Aegilops kotschyi

本研究利用抑制性消减杂交技术,构建了黏类小麦细胞质雄性不育线粒体DNA的消减文库。分别提取相同细胞质背景下的不育和可育等基因系线粒体基因组DNA(mtDNA),用RsaⅠ酶切成大小不等的片段,并各自与不同的接头连接,连续经过两次消减杂交和两次PCR扩增,将PCR产物与克隆载体连接,转化为大肠杆菌感受态细胞DH5α,经蓝白斑筛选后,再用PCR方法插入片段筛选出阳性重组质粒,构建差异DNA消减文库。在构建的不育mtDNA文库中,将SSH文库的全部扩增产物与SSH文库中的单条扩增条带分别进行胶回收和克隆转化,分别挑取54个和6个阳性克隆进行测序分析和相关功能初步比对。结果表明,不育mtDNA的SSH文库差减杂交效率较高,质量较好;回收单条扩增条带分别进行克隆测序的结果准确率较高;对测序序列进行BLASTx比对及功能注释分析发现,约90%的序列来源于线粒体基因nad1和nad5的非编码区。

In this research, we construct subtractive mtDNA library of male sterile lines by employing suppression subtractive hybridization （SSH） technology. Mitochondrial genome DNA were extracted from male sterile and fertile lines with the same cytoplasmic source. After Rsal digestion, tester DNA were divided into two groups to link with the specific adaptor 1 and adaptor 2. Then tester DNA were hybridized with driver DNA twice and underwent nested PCR twice. The PCR products were ligated with pMD19-T vector, transformed into E. coli DH5α, screened through the blue-white screening system, and then the positive recombinant plasmid were identified by PCR approach. mtDNA library was constructed successfully. 54 clones from all the PCR products and 6 clones from single PCR band in the SSH library was cloned respectively, and the positive clones were sequenced and analyzed. The results showed that cDNA libraries constructed was of good quality with high efficiency of SSH, and sequencing results of recycled single PCR bands were of high accuracy. Furthermore, approximately 90% sequences analyzed were derived from non-coding regions of mitochondrial genes including nad1 and nad5 based on BLASTx and functional annotation analysis.