1995 Fiscal Year Final Research Report Summary
MOLECULAR STUDIES ON NUCLEAR GENE (S) INTERACTING RYE CYTOPLASM ORDERING OF YAC CLONES TO IDENTIFY THE GENE (S)
| Project/Area Number |
06660007
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Breeding science
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
MURATA Minoru OKAYAMA UNIVERSITY,RESEARCH INSTITUTE FOR BIORESOURCES,ASSOCIATE PROFESSOR, 資源生物科学研究所, 助教授 (20166292)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Differential screening / Midget chromosome / Nucleo-cytoplasmic interaction / Secale cereale / Triticum aestivum / YAC clones / cDNA / rbcL |
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| Research Abstract |
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.
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