Construction Of Salt Tolorance Engineered Rhizobia And Its Effects On The Growth Of Alfalfa After Inoculation

In recent years, it has been an important research field to modify the genetic characters of rhizobium,by the mean of biotechnological assays, such as nitrogen fixation enzyme, salt tolerance etc. Constructionof engineered rhizobium to improve the salt-tolerance ability of leguminous plants is one of the significantapplication, it will be beneficial to the development of bio-fertilizer in the future.In the present study, HAL1 gene has been isolated form yeast according to the sequence in the publicdatabase. The target gene then was inserted into prokaryotic expression vector pGEX-4T-1 to obtain theengineered alfalfa rhizobium strain by the tri-parental mating. We studied the impact on the inoculatedalfalfa growth and the main results were demonstrated as follows.1, The stability of re-combinated plasmid in the engineered rhizobium. To test the stability of there-combinated plasmid, we sub-cultured the engineered strain in the YMA liquid medium for five timescontinuously under the condition without antibiotic stress, and the results exhibited the plasmid stillmaintain steadily in the host cell. This means that the plasmid could be existed in the rhizobium anddelivered to next generation without loss.2, Extraction and purification of the fusion protein. We extracted and purified the fusion protein from theengineered rhizobium stain using MagneGST^TM protein purification system. The results showed that theprotein product can reach the maximum yield when inducing time was 3h, together with 1.0mmol/l IPTG.The yield of fusion protein was 3mg per 1ml medium which was about 1.56ï¼…of the total soluble protein.3, The tolerance of engineered stain with different salt concentration. Alfalfa wild rhizobium andengineered rhizobium carrying HAL1 gene were incubated in the YMA medium containing 3ï¼…, 5ï¼…, 7ï¼…and 9ï¼…NaCl, respectively. The results demonstrated that each OD₆₀₀ value of engineered stain was alwayshigher than that of wild rhizobium in the YMA with the same salt concentration. It revealed that theengineered strain grow vigorously than the wild one under salt stress..4, Aalfalfa plants growing in the medium with different salt concentration were infected and theinvestigation showed that the growth condition of plants inoculated by engineered strain was better than thecontrol. The control plants died after 30 days whereas the others were still alive. This means the salttolerance of alfalfa plants carrying recombinated plasmid could be improved in some degree.5,The growth condition of alfalfa treated with engineered strain could be promoted. We planed alfalfaplants in the sterilized sand and watered with nitrogen-free nutrition solution regularly. The plants were infected with engineered strain and wild one when the first leaf emerged. The growth condition wasinvestigated 40 days later, and the results indicated alfalfa having engineered plasmid growed better thanthe plants inoculated with wild strain.