MOLECULAR STUDIES ON NUCLEAR GENE (S) INTERACTING RYE CYTOPLASM ORDERING OF YAC CLONES TO IDENTIFY THE GENE (S)

在小麦黑麦细胞质代换系（cereale）-中国春（CS）中，常发现一条额外的端部着丝粒染色体，因其体积小而被称为“侏儒”染色体，这种侏儒染色体从雄性传递受到一定程度的限制。缺少侏儒染色体的种子由于胚乳败育而不能发芽。即使它们发芽，植物也非常脆弱，高度雄性不育。这表明矮染色体起源于黑麦染色体的某个部位（S），并携带黑麦细胞质必需基因（S）。在这项研究中，我试图分离出含有侏儒染色体DNA序列的YAC克隆。虽然大量的克隆被分离，但几乎所有的克隆都被发现是结构修饰的。由于这似乎是由于酵母细胞中的重组而发生的，因此引入了缺乏重组能力的突变株YKK 115（rad^-）。用差异筛选法从（cereale）-CS构建的文库中筛选到黑麦型cDNA，在筛选的20，000个阳性克隆中，有27个与黑麦cDNA杂交的信号比与小麦cDNA杂交的信号强。序列分析表明，大部分克隆来源于黑麦细胞质中的rbcL（RuBisco大亚基）基因。因此，小麦和黑麦的DNA存在多态性。其它的来自cab（叶绿素a/B结合蛋白）基因和rbcS基因，但在小麦和黑麦之间未检测到RFLP。虽然这种差异筛选似乎是有效的分离黑麦型cDNA，cDNA文库应均衡或标准化筛选前，以避免克隆丰富的cDNA，如rbcL。

In the rye cytoplasm substitution line of wheat, (cereale)-Chinese Spring (CS), an extra telocentric chromosomes, which is called "midget" owing to its small size, is always found. Transmission of this midget chromosome is limited to some extent from male side. Seeds lacking the midget chromosome are not germinable because of endosperm abortion. Even if they germinate, the plants are very weak and highly male-sterile. This indicates that the midget chromosome originated from a certain part (S) of rye chromosome and carry an essential gene (S) for rye cytoplasm. In this study, I attempted to isolate YAC clones containing the DNA sequences of the midget chromosomes. Although a large number of the clones were isolated, almost clones were found to be structurally modified. Since this seemed to occur due to the recombination in yeast cells, a mutant strain, YKK115 (rad^-), lacking the ability of recombination was introduced. However, almost no transformed clones were obtained.Differential screening method was also employed to find rye-type cDNAs in a library constructed from (cereale)-CS.Out of 20,000 screened, 27 positive clones showed stronger signals when hybridized with rye cDNAs than with wheat cDNAs. Sequence analysis revealed that almost clones were derived from the rbcL (RuBisco large subunit) gene in rye cytoplasm. Therefore, there were polymorphisms between wheat and rye DNAs. Others were from the cab (chlorophyll a/b binding protein) genes and rbcS genes, but no RFLP were detected between wheat and rye. Although this differential screening seems to be effective to isolate rye-type cDNAs, the cDNA library should be equalized or normalized before screening to avoid cloning abundant cDNAs such as rbcL.