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

在小麦(cereale)-Chinese Spring （CS）的黑麦细胞质代换系中，经常发现一条额外的远心染色体，因其小而被称为“侏儒”。这种小染色体在一定程度上受限于男性侧的遗传。由于胚乳败育，缺少侏儒染色体的种子不能发芽。即使它们发芽了，植株也非常脆弱，雄性不育。说明小染色体起源于黑麦染色体的某一部分，并携带黑麦细胞质必需基因S。在本研究中，我试图分离含有侏儒染色体DNA序列的YAC克隆。虽然大量克隆被分离出来，但几乎都被发现有结构修饰。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YKK115 （rad^-）。然而，几乎没有获得转化的克隆。采用差异筛选法在（谷类）-CS构建的文库中寻找黑麦型cdna。在筛选的2万个克隆中，27个阳性克隆与黑麦dna杂交时的信号强于与小麦dna杂交。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dna之间存在多态性。叶绿素a/b结合蛋白（叶绿素a/b binding protein， cab）基因和红细胞（rbc）基因的RFLP在小麦和黑麦之间均未检测到。虽然这种差异筛选似乎对分离赖氨酸型cDNA是有效的，但在筛选之前应该对cDNA文库进行均衡或标准化，以避免克隆大量的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.