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本工作采用￣３２Ｐ标记的与固氮（ｎｉｆ）和一般氮代谢（ｎｔｒ和ｇｌｎ）系统有关的基因探针，对不同限制性内切酶完全酶解的粪产碱菌Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓ总ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析，证明在粪产碱菌中存在与ｎｉｆＨ、ｎｉｆＤＫ、ｎｉｆＡ、ｎｔｒＡ，ｎｔｒＣ、ｇｌｎＢ、ｇｌｎＤ同源的ＤＮＡ序列。以ＡｚｏｓｐｉｒｉｌｌｕｍｂｒａｓｉｌｅｎｓｅＳｐ７ｎｉｆＡＤＮＡ为探针，经三轮杂交筛选、从粪产碱菌的基因文库获得含有ｎｉｆＡ同源片段的阳性克隆ｐＬＡＦ４３５。将含有ｎｉｆＡ同源序列的６．０ｋｂｌⅡ片段分别亚克隆在ｐＬＡ２９１７和ｐＢｌｕｅｓｃｒｉｐｔＳＫ（ｐＢＳ）载体上，获得亚克隆ｐＬＡＳ６和ｎＢＡＳ６。建立了亚克隆ｐＢＳＡ６中ｎｉｆＡ同源片段的限制性内切酶图谱，利用亚克隆ｐＬＡＳ６的接合转移特性和ｎｉｆＡ突变株功能互补的方法证明，所亚克隆的ＤＮＡ片段具有ｎｉｆＡ功能
In this work, 32P-labeled gene probes related to nif and general nitrogen metabolism (ntr and gln) systems were used for Southern hybridization analysis of Alcaligenes faecalis total DNA of different digestive enzymes It was demonstrated that there are DNA sequences homologous to nifH, nifDK, nifA, ntrA, ntrC, glnB and glnD in Alcaligenes faecalis. Using Azospirillumbrasilense Sp7nifADNA as a probe, three positive clones were screened and the positive clone pLAF435 containing the nifA homology fragment was obtained from the gene library of Alcaligenes faecalis. The 6.0 kblII fragment containing the nifA homology was subcloned into pLA2917 and pBluescriptSK (pBS) vectors, respectively, to obtain subclones pLAS6 and nBAS6. The restriction endonuclease map of the nifA homology fragment in subclone pBSA6 was established. The subcloned DNA fragment was confirmed to have nifA function by utilizing the junctional transfer characteristics of subclone pLAS6 and the function complementary of nifA mutant strain