光合细菌Rhodobacter sphaeroides glnBlacZ 融合子的构建与表达  

Studies on the Construction and Expression of Rhodobacter sphaeroides glnB lacZ Fusion

作　　者：梁莉 朱美珍[1] 吴永强[1] 

机构地区：[1]中国科学院上海植物生理研究所,上海200032

出　　处：《植物生理学报（0257-4829)》1999年第3期249-255,共7页Acta Phytophysiologica Sinica

基　　金：863 计划;国家自然科学基金;上海植物生理所植物分子遗传国家重点实验室资助

摘　　要：亚克隆了Ｒｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ ｇｌｎＢ启动子，以ｐＭＰ２２０ 为载体构建成ｇｌｎＢｌａｃＺ融合子。将ｇｌｎＢｌａｃＺ、ｎｉｆＨｌａｃＺ、ｎｉｆＡｌａｃＺ分别导入Ｒ． ｓｐｈａｅｒｏｉｄｅｓ 谷氨酸合酶突变株ｇｌｔＢ－ 、ｇｌｔＤ－ 和野生型菌株中，分析了突变对固氮基因转录表达的影响。试验证明，在ｇｌｔＢＤ 突变株中ｎｉｆＨ 的表达受阻遏，ｎｉｆＡ 表达水平很低。这证明ｇｌｔ 基因的突变引起固氮酶结构基因和固氮正调节基因的转录被阻遏，而ｇｌｎＢ 基因的表达几乎不受影响。试验还测定了环境中结合态氮和有机酸等信号分子对ｇｌｎＢ 和ｎｉｆＨ 表达的影响，发现加入氨或谷氨酰胺后，ｎｉｆＨ 的表达受到明显的阻遏作用，ｇｌｎＢｌａｃＺ的β半乳糖苷酶活性虽下降３０ ％ 左右但不随结合态氮浓度升高而变化，仍维持在一个较高的水平。α酮戊二酸和丙酮酸对ｎｉｆＨThe cloning and sequencing of glnBA genes of R. sphaeroides strain 6001 were completed as previoursly shown (Han et al . 1996 and 1997). The glnB upstream region containing glnB promoter was subcloned and the glnB lacZ fusion was constructed by using promoter detected plasmid pMP220 as a vector(Fig.1). R. sphaeroides gltD and gltB genes for the small subunit and large subunit, respectively, of glutamate synthase were cloned and mutagenized by employing transposon Tn5 generated mutation in our laboratory (Lu et al . 1996). The fusions of glnB , nifA , and nifH lacZ were mobilized into the gltD - and gltB - mutants as well as the wild type strain 6001 of R. sphaeroides . β galactosidase expression of these fusions were analyzed under different physiological conditions (Table 3). The results indicated that in the wild type strain, the expression of nifA lacZ was at a very low level and almost no expression of nifH lacZ was found and about 70% of β galactosidase expression of glnB lacZ was found in the presence of ammonia. Almost the same level of nifH and nifA lacZ gene expression in the gltD -and gltB - mutants under both nitrogen limited and nitrogen sufficient conditions was observed. Repression of nifA and nifH genes in response to fixed nitrogen was also found in R. sphaeroides gltD - and gltB - mutants. But the mutation of gltD and gltB had no effect on the transcription of glnB gene, since its expression had been shown to be normal in the mutants. Even a rise of the concentration of Gln or ammonium in the medium from 10 to 40 mmol/L did not lower the level of glnB gene expression (Table 4). It was suggested that NH + 4 and glutamine did not act on the glnB product, PⅡ protein, directly. A certain degree of expression of nifH but not glnB was induced by exogenous pyruvate and β ketoglutarate (Table 5).

关 键 词：浑球红细菌 blnB-lacZ 融合子 glt变种 

分 类 号：Q78[生物学—分子生物学] Q945.13