Establishment And Stability Test Of Fluorescence Marked Rhizobiums Based On Triparental Mating

In order to ascertain the nodule occupancy and nodulation rate of target rhizobia,and the competition and growth-decline relations between the target and indigenousrhizobia, tracer technique is needed. Based on classical tri-parental mating (withtaking filter membrane(￠25mm,0.22μm) as carrier), marked rhizobia that containgenetically stable cfp (cyan fluorescent protein) genes were obtained (target rhizobia:Sinorhizobium meliloti (Ensife meliloti) LZgn5, Sinorhizobium meliloti12531,Sinorhizobium meliloti (Ensifer meliloti） LH3436) using different importingconditions, to explore suitable methods for fluorescent plasmid coalesce in rhizobiaand the relating influencing factors, to optimize condition for plasmid coalesce. Theexperiment was set as follows:①. taking filter membrane as carrier, at4℃to mixthalli;②. taking filter membrane as carrier, and inducing the competence of rhizobiaby Ca2+;③. taking alfalfa leaf as carrier to replace filter membrane. Under aboveconditions, conjugation rates and the genetic stabilities of fluorescent plasmid werediscussed. Then respectively inoculated the fluorescence marked rhizobia derivedform S.LZgn5，S.12531and S.LH3436to Gannong No.5alfalfa, measured plantbiomass, root length, plant height, leaf number, leaf area, individual nodulationnumber, nitrogenase activity, nodule occupancy and leaf chlorophyll content ofinoculated alfalfa plants. The results were as follows:1.New screening method was put forward based on the principals that donationand assistant E.coli can not grow on N-free media and cfp can only release cyanfluorescence when combined with Ca2+and been excitated by ultraviolet. TY andN-free medium plus (SM+Gm) plates were used for conjugation screening byalternative culture and verification to remove drug-resistant mutant rhizobia strainsand nutritional deficient reverant of E.coli, and to increase screening and identifyingefficiencies of fluorescence marked rhizobia.2.Carrier had significant effect on the success rate of conjugation. When alfalfaleaf was used as carrier, S.LZgn5, S.12531and S.LH3436all obtained higherconjugation rates, which were significantly （P<0.05）higher than the conjugation rates got with filter membrane as carrier. However, under this treatment, theconjugation rates among different rhizobia also had significant difference (P<0.05),S.LZgn5was the best, and the conjugation rate was8.53×10-6, S.12531was the worst,and the conjugation rate was6.12×10-6.3.Genetic stability of fluorescent plasmid of conjugations obtained with filtermembrane as carrier, and inducing the competence of rhizobia by Ca2+were the worst,in the processing of5to10to20times of subculture, the loss rates of fluorescentplasmid were increasing.4.The fluorescent plasmid of conjugations obtained with alfalfa leaf as carrierhad the best stability. In the processing of5to10to20times of subculture, the loss offluorescent plasmid was not changed.5.The biomass, nitrogenase activity, root length, plant height and leaf chlorophyllcontent of alfalfa plants inoculated with S.LZgn5, S.12531and S.LH3436and theirfluorescence marked strains had significant difference with the treatment of noinoculation, this showed that the inoculation of rhizobia could promote plant biomassaccumulation, and have obvious growth promotion effect.6. Comparing the fluorescence marked rhizobia with their corresponding originalrhizobia, they did no harm to alfalfa plant and could express stably in plant, and hadthe similar growth promotion ability with original rhizobia.