2001 Fiscal Year Final Research Report Summary
Direct cloning of genes for crop cultivation approached by positional Information
| Project/Area Number |
11460005
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Breeding science
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| Research Institution | Kihara Institute for Biological Research, Yokohama City University |
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Principal Investigator |
OGIHATA Yasunari Kihara Institute for Biological Research, Yokohama City University, Associate Professor, 木原生物学研究所, 助教授 (40185533)
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| Project Period (FY) |
1999 – 2001
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| Keywords | Polyploid wheat / Large size DNA / Genomic library / Gene cloning / Q gene / Differential Display / Chromosome deletion lines / Mapping |
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| Research Abstract |
The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method was applied to the cloning of the Q gene of common wheat. Using the AMF method, we comapred the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chines Spring (CS) carrying the Q gene to those of a chloromsome deletion line of CS. Approximately 12200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragmentsw were differentially expressed between CS and deletion line. Northern nad Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genomes. Four clones were genetically mapped to the region deleted. Subsequently, one clone was mapped at the same locus as the Q gene, indicating that the clone corresponds to the Q gene or is tightly linked to it. However, further mapping experiment suggested that the clone was segregated from the Q gene.
Large-insert genomic DNA libraries of common wheat were constructed in a newly developsed transformation-competent artificial chromosome (TAC) vector, which accepts and maintains large genomic DNA fragments stably in both Escerichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. TAC clones containing genes(s) of interest were immediately transferred into grass genomes with the Agjobacterium system. TAC libraries constructed canbe used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.
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