Investigation of endophytic N_2-fixers by PCR method

从不同品种、不同地点、不同采样时间的甘蔗茎叶样品中提取基因组DNA，利用巢式pcr扩增nifH序列片段。与慢生根瘤菌和克雷伯菌的nifH基因高度相似的DNA序列在不同的采样条件下都经常被发现。通过巢式pcr和rt巢式pcr分析，从盆栽栽培的甘蔗根和茎中扩增出与慢生根瘤菌nifH基因高度相似的DNA序列，结果表明表达了与慢生根瘤菌nifH基因同源的DNA序列。这些与慢生根瘤菌nifH基因同源的序列显示出多样性，说明内生慢生根瘤菌在甘蔗中具有固氮作用。另一方面，在大田甘蔗茎中表达了与梭状芽胞杆菌和克雷伯氏菌同源的nifH基因。在大田菠萝叶片中检测到与偶氮菌和黄杆菌同源的nifH基因的表达。从大田红薯的叶、叶柄、茎和块茎中提取基因组DNA和RNA。采用巢式pcr方法，用两组简并引物扩增nifH基因片段。在叶柄中发现了与日本慢生根瘤菌和固氮小芽孢杆菌nifH基因高度相似的DNA序列，在茎中发现了与变水芽孢菌（Anabaena variabilis）、seropedicae、A. 7120和Nostoc commune基因相似的DNA序列，在块茎中发现了与豆科根瘤菌（leguminosarum）和日本根瘤菌（B. japonicum）基因相似的DNA序列。RT-nested-PCR分析显示，茎中表达了缓生根瘤菌和未培养的n_2固定菌的同源克隆，块茎中表达了未培养的n_2固定菌的同源克隆。对甘薯DNA和RNA扩增产物的序列分析表明，甘薯组织中存在多种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.