2005 Fiscal Year Final Research Report Summary
Investigation of endophytic N_2-fixers by PCR method
| Project/Area Number |
15380047
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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 |
Plant nutrition/Soil science
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YONEYAMA Tadakastu The University of Tokyo, Graduate School of Agricultural and Life Sciences, Professor, 大学院・農学生命科学研究科, 教授 (70124360)
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| Co-Investigator(Kenkyū-buntansha) |
ANDO Shotaro National Institute of Livestock and Grassland Science, Department of Grassland Ecology, Chief Researcher, 畜産草地研究所, 研究室長 (00370578)
OHWAKI Yoshinari National Agricultural Research Center, Department of Soil and Fertilizer, Senior Researcher, 畜産草地研究所, 主任研究官 (60355542)
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| Project Period (FY) |
2003 – 2005
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| Keywords | Biological Nitrogen Fixation / Endophytes / Sugarcane / Sweet potato / Pineapple / N_2-fixers / nifH / PCR |
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| Research Abstract |
Genomic DNA was extracted from stem and leaf of sugarcane sample with different varieties, location and sampling period and the fragment of nifH sequence was amplified by nested-PCR. The DNA sequences with high similarity to the nifH gene of Bradyrhizobium and Klebsiella were frequently found irrespective of sampling condition. From root and stem of sugarcane, which was grown in a pot experiment, the DNA sequences with high similarity to the nifH gene of Bradyrhizobium were amplified by nested-PCR and RT-nested-PCR analysis revealed that the nifH genes homologous to those of Bradyrhizobium were expressed. These sequences homologous to the nifH gene of Bradyrhizobium showed diversity and suggested that endophytic Bradyrhizobium fix nitrogen in sugarcane. On the other hand, the nifH genes homologous to those of Clostridium and Klebsiella were expressed in stem of field-grown sugarcane. The expression of the nifH genes homologous to those of Azoarcus and Xanthobacter were detected in leaf of field-grown pineapple.
Genomic DNA and RNA from the leaf, petiole, stem and tuber samples of field-grown sweet potato were extracted. The fragments of nifH gene were amplified with two sets of degenerate primers by nested-PCR. The DNA sequence with high similarity to the nifH gene from Bradyrhizobium japonicum and Paenibacillus azotofixans were found in petiole, Anabaena variabilis, Herbaspirillum seropedicae, A. 7120 and Nostoc commune were found in stem, and Rhizobium leguminosarum and B. japonicum were found in tuber. RT-nested-PCR analysis revealed that the clones homologous to Bradyrhizobium sp.and uncultured N_2-fixing bacterium were expressd in stems, and clones homologous to uncultured N_2-fixing bacterium was expressd in tuber. Sequence analysis of the PCR products amplified from DNA and RNA isolated from sweet potato revealed that the existence and expression of diverse nifH sequences in sweet potato tissue.
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