Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

温敏核不育水稻5460S细菌人工染色体文库的构建和鉴定

为了构建温敏核不育 (TGMS)基因区域的精细物理图谱并最终分离TGMS基因 ,以温敏核不育水稻 5 46 0S为材料 ,摸索优化了构建植物细菌人工染色体 (BAC)文库的方法 ,构建了一个高质量的BAC文库 .该文库由 1 95 84个克隆构成 ,插入片段平均长度为 1 1 0kb ,相当于水稻单倍体基因组大小的 5倍 ;以分子量分别为 1 40和2 5 0kb的 2个大BAC克隆进行稳定性传代实验 ,经 1 0 0代传代后其插入的DNA片段仍然稳定存在 ;以线粒体和叶绿体基因为探针筛选BAC文库 ,未检验出叶绿体和线粒体DNA的污染 ;以和tms1基因连锁的 3个分子标记作为探针对BAC文库进行了筛选 ,每个探针至少可获得一个阳性克隆 ,利用热不对称性交错PCR(Tail PCR)法成功分离了阳性克隆的左右末端序列 .     

普通小麦三种细胞质雄性不育系线粒体DNA的比较研究

对细胞质分别来源于粘果山羊草（Ａｅ．ｋｏｔｓｃｈｙｉ）、偏凸山羊草（Ａｅ．ｖｅｎｔｒｉｃｏｓａ）、提莫菲维（Ｔ．ｔｉｍｏｐｈｅｅｖｉ）的３种普通小麦雄性不育系,其相应保持系和共有的一种恢复系的ｍｔＤＮＡ进行了ＲＦＬＰ比较分析。发现Ｋ型和Ｖ型不育系的ｍｔＤＮＡ在组织结构上不同于Ｔ型,说明Ｋ、Ｖ型不育系是有别于Ｔ型的两种新不育类型。Ｋ型、Ｖ型不育系的ｍｔＤＮＡ与保持系和恢复系显著不同,推测ｍｔＤＮＡ与小麦细胞质雄性不育性有关。实验同时发现Ｔ型不育系与其保持系的ｍｔＤＮＡ非常相似,对这种相似性的原因进行了讨论。     

Molecular characterization of a new type of cytoplasmic male sterile sugar beet

A line (named Cl) of cytoplasmic sterility of sugar beet whose cytoplasm derived from Betacicla Turkey was obtained by interspecific hybrid. Its cytoplasm and a spontaneous male sterile cytoplasm from wild beet Beta maritima (named M) were compared with that of Owen's sterile line (S-cms) and a common maintainer of them named N was used as control. RFLP and RAPD methods were mainly used in our experiments. The restriction fragment patterns of mtDNAs were found to be likely but for a few of specific low-lighted electrophoresis bands in Cl. The results of Southern hybridization of six heterogeneous mitochondrial genes as probes to digests of mtDNAs by six restriction enzymes showed to be analogous between S and M lines. But the Cl mtDNA was sorted out by hybridization of atpA probe. Difference of low-molecular-weight mitochondrial DNAs was found among the three sterile lines. Three RNA molecules weighing about 4.2kb stably existed in Cl mitochondria. Our results of RAPD also supported that the Cl cytoplas     

一种新型甜菜胞质雄性不育系的分子生物学鉴定

对通过种间杂交获得的两种胞质雄性不育系甜菜(命名为Cl和M,其细胞质来源分别为Beta cicla Turkey和Beta maritima)和通常的Owen型胞质不育系甜菜(简称作S)以及这3种不育系通用的保持系(简称N)进行了线粒体基因组的RFLP和RAPD对比分析.结果表明Cl与S相比线粒体组织结构具有明显的差别.用6种异源线粒体基因作探针对6种限制性内切酶片段进行Southern杂交分析,发现Cl和S线粒体基因组之间存在明显多态性.但S和M之间却异乎寻常地表现一致.RAPD分析验证了Cl和 S之间的差异,此外,在Cl中发现 3种 4.2 kb大小的特异存在的RNA分子.根据结果认为Cl是一种不同于S的新型细胞质不育系.用RAPD方法也找到了S与M之间差异.酶切结果表明N与3个不育系的mtDNA非常相似.但在杂交和RAPD分析中均找到了N系的特异片段.这些片段是否与育性相关尚有待分析.