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Investigation of endophytic N_2-fixers by PCR method

PCR法研究内生N_2-固定菌

基本信息

批准号：
15380047
负责人：
YONEYAMA Tadakastu
金额：
$ 9.86万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15380047/
关键词：
Biological Nitrogen Fixation Endophytes Sugarcane Sweet potato Pineapple N_2-fixers nifH PCR 窒素固定菌

项目摘要

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.

以不同品种、不同产地、不同取样时期的甘蔗茎、叶为材料，提取基因组DNA，采用巢式PCR扩增nifH基因片段。与慢生根瘤菌和克雷伯氏菌的nifH基因具有高度相似性的DNA序列经常被发现，无论采样条件如何。以盆栽甘蔗的根和茎为材料，通过nested-PCR扩增出与慢生根瘤菌nifH基因高度同源的DNA序列，RT-nested-PCR分析表明，与慢生根瘤菌nifH基因同源的基因均得到了表达。这些序列与甘蔗慢生根瘤菌nifH基因同源，具有多样性，表明甘蔗内生慢生根瘤菌具有固氮作用。另一方面，与梭菌和克雷伯氏菌同源的nifH基因在田间生长的甘蔗茎中表达。从田间种植的菠萝叶片、甘薯叶柄、茎和块茎中提取基因组DNA和RNA，检测到与Azoarcus和Xanthocarpus同源的nifH基因在菠萝叶片中的表达。用两对简并引物对nifH基因进行巢式PCR扩增。与慢生根瘤菌和固氮类芽孢杆菌的nifH基因高度相似的DNA序列存在于叶柄、变异鱼腥藻、血清草螺菌、A. 7120和地木耳，根瘤菌和B.在块茎中发现了日本根瘤菌。RT-nested-PCR分析表明，与慢生根瘤菌和未培养固氮菌同源的克隆在茎中表达，与未培养固氮菌同源的克隆在块茎中表达。从甘薯中提取的DNA和RNA经PCR扩增后的产物进行序列分析，结果表明甘薯组织中存在不同的nifH基因序列并表达。

项目成果

期刊论文数量（25）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

エンドファイテイック窒素固定第三タイプの植物-微生物窒素固定システム

内生固氮第三种植物-微生物固氮系统

DOI：
发表时间：
2005
期刊：
化学と生物 43・12
影响因子：
0
作者：
安藤象太郎;大脇良成;後藤匡裕;米山忠克
通讯作者：
米山忠克

Contamination of nif-like DNA in PCR reagent

PCR试剂中nif样DNA的污染

DOI：
发表时间：
2005
期刊：
Abstracts of the 15th annual meeting of Japanese Society of Plant Microbe Interac.
影响因子：
0
作者：
M.Goto;Y.Hachisuka;S.Ando;T.Yoneyama
通讯作者：
T.Yoneyama

Role of nitrogen fixation for sustainable sugarcane production.

固氮对可持续甘蔗生产的作用。

DOI：
发表时间：
2004
期刊：
Proceedings of the International Symposium on Sustaiabe Sugarcane and Sugar Production Technology.
影响因子：
0
作者：
S.Ando;S.Meunchang;S.Thippayarugs;P.Prasertsak;N.Matsumoto;T.Yoneyama
通讯作者：
T.Yoneyama

大脇良成, 高畑康紀, 米山忠克, 藤原伸介: "重窒素自然存在比法および重窒素同位体希釈法を用いたエンドファイトによるサツマイモの窒素固定量の推定"植物微生物研究会第13回研究交流会講演要旨集. (印刷中). (2004)

大胁义成、高畑康典、米山忠胜、藤原伸介：《利用重氮自然丰度比法和重氮同位素稀释法估算红薯内生菌的固氮量》植物微生物研究组第13次研究交流会文集讲座摘要（正在出版）（2004）。