Selbitschka W, Jording D, Nieman S, Schmidt R, Pühler A, Mendum T, Hirsch P
Lehrstuhl für Genetik, Universität Bielefeld, Germany.
FEMS Microbiol Lett. 1995 May 15;128(3):255-63. doi: 10.1111/j.1574-6968.1995.tb07533.x.
An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II (nptII) promoter of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.
构建了一种整合载体，可将克隆的DNA插入豌豆根瘤菌蚕豆生物变种染色体的非必需位点。整合基因的表达受转座子Tn5组成型新霉素磷酸转移酶II（nptII）启动子的控制。该载体的设计确保能够检测到载体序列的缺失，从而能够选择仅含有所需DNA的后代。使用新构建的载体将赋予GUS活性的大肠杆菌gusA基因插入已消除其共生质粒（pSym）的豌豆根瘤菌蚕豆生物变种菌株LRS39401中。一个GUS阳性的接合子菌株CT0370被证明已丢失所有载体序列。将pSym2004（共生质粒pRL1JI的Tn5标记衍生物，可指定豌豆结瘤和共生固氮）接合转移到CT0370，恢复了该GUS阳性菌株的共生能力。目前，CT0370菌株正在用于田间释放试验，以评估在环境条件下天然豌豆根瘤菌蚕豆生物变种群体中pSym转移的程度。
