巴西固氮螺菌Yu62draTG基因及其下游区域的克隆与核苷酸序列分析  

Cloning and Sequencing of draTG Genes and Their Downstream Region of Azospirllum brasilense Yu62

作　　者：马旅雁[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》1997年第3期227-235,共9页Chinese Journal of Biotechnology

基　　金：863计划资助项目

摘　　要：以ＡｚｏｓｐｉｒｉｌｕｍｂｒａｓｉｌｅｎｓｅＳｐ７４．０ｋｂｄｒａＴＧ片段为探针，自Ａ．ｂｒａｓｉｌｅｎｓｅＹｕ６２的基因文库中克隆了约８ｋｂ的ｄｒａＴＧ同源片段。通过对该片段的Ｓｏｕｔｈｅｒｎ杂交分析发现Ａ．ｂｒａｓｉｌｅｎｓｅＹｕ６２的ｄｒａＴＧ基因定位在３０ｋｂＥｃｏＲＩＫｐｎＩ片段上，其上游与ｎｉｆＨ基因相邻。ＤＮＡ序列分析结果表明：该片段含有完整的ｄｒａＴＧ，这两个基因下游还有两个开放阅读框架（ＯＲＦ３和ＯＲＦ４，其中ＯＲＦ４是不完整的），ｄｒａＴＧ及下游的ＯＲＦ３推测以一个操纵元的方式转录；在ｄｒａＧ及ＯＲＦ３的上游区域均发现σ５４依赖型启动子的特征序列（ＤＰＥ及ＵＡＳ），推测它们与ｄｒａＴ共转录外，还有可能单独转录。同源比较的结果表明Ａｚｏｓｐｉｒｉｌｕｍ的ＤｒａＴＧ是非常保守的，它们在菌株及种间的差异都很小；紧接着ｄｒａＧ的ＯＲＦ３除与Ａ．ｌｉｐｏｆｅｒｕｍ和Ｒｈｏｄｏｓｐｉｒｉｌｕｍｒｕｂｒｕｍ相应位置的ＯＲＦ同源外，还与Ａｚｏｔｏｂａｃｔｅｒｖｉｎｅｌａｎｄｉ的ＯＲＦ１４同源；A 8kb fragment was cloned by probing the gene library of Azospirillum brasilense Yu62 with the 4.0kb draTG fragment of A.brasilense Sp7.DNA hybridization of this fragment demonstrated that draTG genes were located in a 3.0kb EcoRⅠ-KpnⅠ fragment,and contiguous to nifH gene.This 3.0kb fragment was completely sequenced on both strands.Sequence analysis of the fragment revealed that it included the full lenghth draTG genes and two ORF downstream of draG (ORF3 and ORF4,ORF4 is incomplete).The draTG and downstream ORF3 are assumed to be co transcribed as a single operon.Promoter element analysis of the sequenced region showed that there were some elements of σ 54 dependent promoter (DPEs and UASs) in upstream region of draG and ORF3.This suggests that the draG and ORF3 might tbe transcribed independently except for being cotranscribed with draT.Both the DNA and amino acid sequences of the draTG genes from A.brasilense Yu62 were compared with those of other nitrogen fixing bacteria.The results showed the DraTG were highly conservative.There were only few changes among strains and/or species.Homology analysis of ORF revealed that the ORF3 immediately downstream of draG was homologous not only with the ORF at the same position in Rhodospirillum rubrum and A.lipoferum ,but also with the ORF14 of Azotobacter vinelandii ,and the ORF4 showed extensive similarity to yafJ of Escherichia coli.

关 键 词：巴西固氮螺菌 基因 克隆 核苷酸序列 

分 类 号：Q939.113[生物学—微生物学]