巴西固氮螺菌Yu62 draTG基因及其下游区域的定位诱变分析  

Site-directed Mutagenesis Analysis of draTG Genes and Their Downstream Region of Azospirillum brasilense Yu62Project of Chinese National Programs for High Technology Research and Development(No.863 101 03 04 02).

作　　者：马旅雁[1] 吴粤[1] 王娟 赵银锁[1] 李季伦[1] 

机构地区：[1]北京农业大学农业生物技术国家重点实验室

出　　处：《生物工程学报》1999年第3期281-287,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划项目

摘　　要：用卡那霉素盒（Ｋｍｒｃａｓｅｔｅ）插入法，对巴西固氮螺菌（Ａｚｏｓｐｉｒｉｌｕｍｂｒａｓｉｌｅｎｓｅ）Ｙｕ６２的ｄｒａＴＧ基因及其下游区域进行了诱变，并获得相应的突变株。研究表明：ｄｒａＴ变突株的固氮酶活性不再受铵抑制，而ｄｒａＧ突变株在有铵时则丧失固氮酶活性，但当铵耗尽后却不能像野生型菌株那样恢复活性。ｄｒａＴＧ下游区域突变株ＹＺ４（突变位点距ｄｒａＧ约２ｋｂ）在无氮及限铵条件下，其固氮酶活性比野生型菌株的高，但其ｎｉｆＨｌａｃＺ转录融合子的表达并不受影响。draT and draG genes are involved in post translational regulation of nitogenase activity of Azospirillum brasilense Yu62.Both genes and their downstream region were mutagenized by Km r cassette insertions. Analysis of mutations introduced into the dra region on the A.brasilense Yu62 chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium.In contrast,a mutant with an insertion in draG was still capable of ADP ribosylating dinitrogenase reductase in response to ammonium, but was no longer able to recover the activity after ammonium depletion. These results confirm that draTG genes are active in the regulation of nitrogenase activity of A.brasilense Yu62. Analysis of mutations introduced into the draTG downstream region (the mutagenized site is about 2kb downsteam from draG ) showed that the nitrogenase activity of the mutants were higher than that of the wild strain while growing in nitrogen free medium and medium with 2 mmol/L ammonium.These results reveal that there isn't any gene that is necessary for transcription of nifHDK in the mutagenized region,but it is possible that there are some genes which play a role in regulation of the activity of nitrogenase. The results of monitoring the expression of transcriptional nifH lacZ gene fusion in the mutant YZ4 showed that the transcription of nifH in the mutant YZ4 was the same as that in wild strain Yu62.

关 键 词：巴西固氮螺菌 draTG 突变株 定位诱变 

分 类 号：Q939.113[生物学—微生物学]