摘要
合成玉米CMS S线粒体DNAR区域端点和orf77两端的特异引物 ,对Mo17、W2 3、77为核背景的N、C、T、S等 4种胞质的 2 6个CMS材料的线粒体DNA进行了PCR扩增。结果表明orf77和R区域具有典型的胞质特异性 :S组中有完整的R区域和orf77;T组中有部分R区域 ,可以扩增出orf77的对应片段 ,但其核苷酸序列与S组orf77不同 ;正常胞质 (N)中有部分R区域 ,但不包含完整的orf77;C胞质中不包含R区域。结果表明orf77是玉米S组CMS特异的胞质育性候选基因。在进行连锁群步移的基础上 ,建立了基于BAC文库、双酶切和Southern杂交的构建线粒体DNA物理图谱的体系。绘制了Mo17CMS J线粒体DNAR区的部分物理图谱。理论上 。
Total DNA from twenty six CMS lines of maize under Mo17,77 and W23 nuclear background were used for PCR amplification, including N, T, C, S four groups of cytoplasms. The primers was prefabricated according the sequence of R region published by Zebala (1997). Through these amplifications, mitochondrial DNA fragments were obtained from maize total DNA. Generally the results in one group are identical. And they are different from the others. The amplified fragments were sequenced and also give us much more information about the structure of mitochondrial genes that may lead to CMS. In order to isolate and identify the CMS genes, we developed a new platform to construct physical map of chromosome DNA by means of restriction enzyme double digestion. The elongation of contigs is based on Southern hybridization. Having retrieved DNA from agarose gel after electrophoresis by β agarase, we labelled it with 32 P dCTP as a probe. We detected the positive clones in the gene library. Two contigs were revealed. And a restriction map covering 40kb was constructed, including R region.
基金
国家自然科学基金 (3 0 0 70 475 )
高校博士点基金项目 (980 40 4)
农业部"948"项目 (2 0 10 10 )
