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采用 PCR 技术,获得含粪产碱菌(Alcaligenes faecalis)nifH 基因启动子的0.6 kbHind Ⅲ-BamHI 的扩增片段,核苷酸序列分析结果表明,该 PCR 产物的序列与已报道的模板 nifH启动子序列完全一致。通过两轮基因连接、转化和筛选,获得 nifH 启动子与 gusA 正向连接的融合基因表达载体 pTGY7。经三亲结合将 pTGY7导入粪产碱菌中,证明该融合基因能在结合子中正常表达。采用融合基因表达的组织化学定位方法,研究粪产碱菌在根表和根内的定殖和 nifH 基因的表达,结果表明在低铵浓度下,粪产碱菌能紧密地结合在水稻根表,部分菌体能侵入根内,nifH::gusA 融合基因在根际联合固氮体系中能高水平表达。
The amplified fragment of 0.6 kb Hind III-BamHI containing the promoter of nifH gene of Alcaligenes faecalis was obtained by PCR. The nucleotide sequence analysis showed that the sequence of the PCR product was identical to the reported template nifH promoter The sequence is exactly the same. Through two rounds of gene connection, transformation and selection, a fusion gene expression vector pTGY7 with nifH promoter and gusA forward connection was obtained. The pTGY7 was introduced into Alcaligenes faecalis via triadic conjugation, which proves that the fusion gene can be expressed normally in the binders. The colonization and nifH gene expression of Alcaligenes faecalis in the root and root were studied by using the method of histochemical localization of fusion gene. The results showed that Alcaligenes faecalis could bind closely to the root surface of rice at low ammonium concentration , Part of the bacteria can invade the root, nifH :: gusA fusion gene in the rhizosphere nitrogen fixation system can be high-level expression.