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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 study, Suppression subtractive hybridization (SSH) technique was used to construct a subtractive library of mitochondrial DNA in the cytoplasmic male sterile line of wheat. Mitochondrial genomic DNA (mtDNA) was extracted from sterile and fertile isogenic lines with the same cytoplasmic background, respectively. The mtDNA was digested with RsaI into fragments of different sizes and each was ligated with different linkers. After two successive subtractive hybridizations and two After PCR amplification, the PCR product was ligated with the cloning vector and transformed into E. coli competent cells DH5α. After being screened by blue and white spots, the recombinant plasmid was screened by PCR and inserted into the fragment to construct a subtractive DNA subtractive library. In the constructed sterile mtDNA library, all the amplified products of the SSH library and the single amplified bands in the SSH library were respectively subjected to gel recovery and cloning transformation. 54 and 6 positive clones were picked for sequencing analysis and correlation Function of the initial comparison. The results showed that the subtractive hybridization efficiency of SSH library of sterile mtDNA was high and its quality was good. The accuracy of cloning and sequencing of single amplified bands was high. By BLASTx alignment and functional annotation analysis, About 90% of the sequences are derived from the non-coding regions of the mitochondrial genes nad1 and nad5.