质粒exoR′恢复紫云英根瘤菌胞外多糖缺陷型变种有效结瘤能力

Restoring the Symbiotic Deficiency of the Exo-Mutants on Astragalus Sinicus by Plasmid exoR'

以ＰＭＮ２作为载体质粒，应用体内克隆技术，从紫云英根瘤菌胞外多糖缺陷型变种ＮＡ－１１中，分离到ｅｘｏＲ′质粒，该杂合质粒带有Ｔｎ５及其插入位点两侧的根瘤菌ＤＮＡ片段。ｅｘｏＲ′质粒能稳定地存在于紫云英根瘤菌中，不仅可纠正紫云英根瘤菌Ｅｘｏ－变种（Ｎｄｖ－）的胞外多糖合成缺陷，也能恢复该变种使宿生植物根部结瘤的能力。

Plasmid pMN2, a Kms derivativeof R68. 45, was used to construct R-prime plasmids carrying Rhizobiun DNAregions coding for exopolysaccharidesynthesis. After pMN2 was introducedinto the Exo-mutunts, a purified Tortransconjugant from each mating waschosen as a donor to mate with Escherichia coli recA-strain HB101, andthe transfer of the Kmr marker oftransposon Tn5 was selected. Kmrcolonies arose at a frequency of about10-7 Per recipient. All repurified Kmrcolonies were found to be TorCbr(pNN2 marker). Test of co-transferfrequency of Kmr of Tn5 showed thatit was genetically linked to vecter plasmid pMN2. Plasmid visualization using a modified Eckhardt techniqueshowed that the size of the plasmidspresent in these transconjugants wasmuch greater than that in pMN2. Thusthese plasmids were considered to carryTn5-flanking Rhizobium DNA sequencesand were named R-prime plasmids.One of such exoR' plasmids derivedfrom Exo-mutunt, NA-11, was ableto fully complement two mutunts,NA-01 and NA-02, and Partiallycomplement the mutunt NA-04. Allthe Exo+ transconjugants induced Fix+nodules as did the wild-type strain.The inoculum reclined its mucoid morphology after being re-isolated fromthe nodules, and the section of thesenodules showed an extensive bacteroidzone containing rod-shaped bacteria,as well as infection threads Penetratingthe root tissue and infecting plant cells.These results demonstrated that complementution of these mutunts byexoR' also restored the ability of symbiotic nitrogen fixation on the plant.