Upadhyaya N M, Scott K F, Tucker W T, Watson J M, Dart P J
Plant Molecular Biology Group, Research School of Biological Sciences, Australian National University, A.C.T.
Mol Plant Microbe Interact. 1992 Mar-Apr;5(2):129-43. doi: 10.1094/mpmi-5-129.
Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.
根瘤菌菌株IC3342结瘤会在某些热带豆科植物如木豆（Cajanus cajan）中引发叶片卷曲综合征（N.M. 乌帕德亚亚、J.V.D.K. 库马尔·拉奥、D.S. 莱瑟姆和P.J. 达特，《植物生理与分子病理学》39:357 - 373，1991）。对这种诱导叶片卷曲（Curl +）的根瘤菌菌株进行转座子（Tn5）诱变，产生了两个Curl - Fix - 和三个Curl - Fix + 突变体。用Tn5、nif和nod基因探针进行质粒可视化及后续的Southern杂交分析表明，在三个突变体中Tn5元件已插入共生（Sym）质粒。限制性内切酶分析表明，没有一个Tn5插入是紧密连锁的。从每个突变体中克隆含Tn5的EcoRI片段，并用作探针从黏粒（pLAFR3）基因组文库中分离相应的野生型DNA片段。通过引入含相应野生型DNA的黏粒，每个突变体中Fix + 和/或Curl + 表型得以恢复。Southern杂交显示，一种密切相关但Curl - 的根瘤菌菌株ANU240含有除根瘤菌菌株IC3342一个已鉴定遗传区域外的所有保守DNA序列。含有IC3342菌株特有的遗传区域（命名为lcr1）的黏粒赋予菌株ANU240 Curl + 表型。对克隆的lcr1区域进行DNA序列分析，揭示了五个开放阅读框（ORF）。ORF2与大肠杆菌调节基因ompR具有同源性，ORF4分别与大肠杆菌和苜蓿根瘤菌调节基因fnr和fixK具有同源性。
