Construction Of Salt-tolorence Gene-engineered Rhizobia And Evaluate Transformant's Physiological Characteristics

To alleviate the dilemma of the outstanding nitrogen-fixation bradyrhizobia with low salt-tolerance ability and these resources can not be widely exploited in large saline area of China, in this study the bradyrhizobia strains of superior application potential which came from two important leguminous plants of soybean and peanut were selected as receptors. Using gene-engineering techniques, two genes with close relation to salt-tolerance—betH(which encodes the transport protein of glycine betaine) and betAB(which encodes the synthesizing protein of glycine betaine) were transducted into soybean and peanut bradyrhizobia to construct salt-tolerance strains, respectively. Furthermore, the expression activities of the exotic genes of transformant strains were analyzed and the physiology characteristics of the gene-modified strains were also tested. The main results as follows:1. betH gene was transducted in bradyrhizobia and 354 transformant strains were obtained. Among them, 4 strains' betH gene was demonstrated existing by plasmid and the other 350 engineered-strains' betH gene was inserted in genome. Via screening on NaCl added plates, two gene-operated bradyrhizobium strains(2764/ pSZ1 .5, 110/ pSZ1 .5) were screened out with the increased salt-tolerance ability of 50%, that means their salt-tolerance concentration was improved from 80mM to 120mM.2. betAB gene was transducted in bradyrhizobia and two transformant strains were obtained. The analysis revealed that the transducted betAB gene existing in the form of plasmid. Tested on NaCl added plates, one gene-operated bradyrhizobium (2764/ betAB) was screened out with its increased salt-tolerance ability of NaCl concentration from 80mM to 120mM.3. The analysis for the express of the two transducted genes revealed that engineering strains of rhizobium that gene was taked by plasmid in rhizobium, Via input the NaCl into the culture medium, screen out 1 engineering strain of rhizobium which increase the Salt Tolorence 50%(from 80mM to 120mM)(2764/betAB) .4.Salt-tolerance ability of the gene-engineered strain 2764/betAB is positive correlation to its glycine betaine content which is 3.2 times comparing to the native strain. While the glycine betaine's content in engineered strains of 2764/ pSZ1.5 and 110/ pSZ1.5 has no distinct change. Further testing discovered that the content of 5 kinds of amino acid of the two gene-engineered strains increased greatly, which suggested the increase has relationship with its enhanced salt-tolerance ability.5.By analyzing the physiological characteristics of the gene-engineered strains , results showed that: (1)Generation time of the gene-engineered strains were 1-2 hours longer than the primary strains;(2)Drought resistance of the gene-engineered strains was weaker than or almost be equal to that of primary strains, which implicated that the ability of drought-resistance had no direct correlation with the transducted genes;(3) Thegene-engineered strains 2764/ pSZ1.5 and 110/ pSZ1.5 were very steadily because the gene in them were inserted in the genome while the exotic gene in strain 2764/betAB had relative weak stability;(4)Nodulation experiments showed that gene-engineered strains of 2764/ pSZ1.5 and 2764/betAB can modulate normally even in lOOmM NaCl condition while the primary strain can not nodulate at the same NaCl concentration. The soybean gene-engineered bradyrhizobium of 110/ pSZ1.5 can not modulate normally at lOOmM NaCl condition or in the condition of no osmotic potential.