1995 Fiscal Year Final Research Report Summary
ABSTRACTS OF RESEARCH PROJECT,GRANT-IN-AID FOR SCIENTIFIC RESEARCH (1995)
| Project/Area Number |
06660426
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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 |
Applied molecular and cellular biology
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| Research Institution | Nara Institute of Science and Technology |
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Principal Investigator |
HASHIMOTO Takashi School of Biological Sciences Associate Professor, バイオサイエンス研究科, 助教授 (80180826)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Yasuyuki School of Biological Sciences Professor, バイオサイエンス研究科, 教授 (50026415)
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| Project Period (FY) |
1994 – 1995
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| Keywords | tobacco / nicotine / mutant / putrescine / molecular biology |
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| Research Abstract |
Two semi-dominant loci, Nic1 and Nic2, regulate nicotine contents in tobacco. We previously isolated two classes of cDNA clones that were under-represented in low-nicotine nic1 nic2 mutant tobacco roots. One of the two cDNA classes was found to encode putrescine N-methyltransferase (PMT) , the first enzyme in nicotine biosynthetic pathway. In this study, we isolated the PMT gene as the first step to unravel the regulatory mechanism of nicotine biosynthesis by Nic genes.
From a Nicotiana sylvestris genomic library, we isolated five genomic clones that hybridized to the PMT cDNA probe, which were divided into three groups according to restriction maps. Genomic Southern hybridization analysis also indicated that the genome of N.sylvestris contains three DNA sequences that are homologous to PMT.
3'-Nontranslated region of the PMT cDNA hybridized only one group in the three PMT-related groups. Northen hybridization with the same PMT-specific probe detected a signal only in the root of N.sylvestris. The group was designated as the PMT gene and further analyzed. The nine exons have very high similarity to the PMT cDNA,including the 3'-nontranslated region described above. A TATA-related region was found 113 bp upstream of the first ATG.We also constructed plant vinary vectors in which the 5'-upstream regions were fused translationally to a reporter gene.
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