Investigation of endophytic N_2-fixers by PCR method

从不同品种、不同地点、不同取样时期的甘蔗样品茎叶中提取基因组DNA，用套式聚合酶链式反应扩增出nifH序列片段。无论采样条件如何，与慢生根瘤菌和克雷伯氏菌nifH基因高度相似的DNA序列经常被发现。从盆栽甘蔗的根和茎中扩增出与慢生根瘤菌nifH基因高度相似的DNA序列，RT-巢式聚合酶链式反应分析表明，与慢生根瘤菌同源的nifH基因在甘蔗中得到了表达。这些与慢生根瘤菌nifH基因同源的序列表现出多样性，表明内生慢生根瘤菌固定甘蔗体内的氮素。另一方面，与梭状芽孢杆菌和克雷伯氏菌同源的nifH基因在田间甘蔗茎中表达。在田间种植的菠萝叶片中检测到与固氮菌和黄杆菌同源的nifH基因的表达，提取了田间种植的甘薯叶片、叶柄、茎和块茎样品的基因组DNA和RNA。用两套简并引物对nifH基因片段进行套式聚合酶链式反应。在叶柄中发现了与日本慢生根瘤菌和固氮拟青杆菌nifH基因高度相似的DNA序列，在茎中发现了变异鱼腥藻、血清型草本菌、A.7120和发菜，在块茎中发现了豆根瘤菌和日本根瘤菌。RT-Nest-PCR分析表明，与慢生根瘤菌同源的克隆和未培养的固氮菌在茎中表达，而与未培养的固氮菌同源的克隆在块茎中表达。从甘薯分离的DNA和RNA中扩增出的扩增产物进行了序列分析，结果表明甘薯组织中存在不同的nifH序列并表达。

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.