Study Of GFP-tagged Phosphorous Solubilizing Bacteria In Grape Rhizosphere Colonization

Phosphorus has great significance in plant growth and development. Under normalcircumstances, the available phosphorus content in the soil is relatively low; most of those arethe invalid phosphorus which is difficult for the plant to use directly. The growing number ofstudies has shown that rhizosphere microorganisms on the soil properties have a significantimpact to the physiological activities of plant roots, plant nutrient uptake, plant growth anddevelopment. Rhizospheric microorganism can play a good effect to reduce the use ofinorganic phosphate in the process of intensive agricultural development, change the insolublephosphate in the soil to available phosphorus content, and is conducive to the sustainabledevelopment of agriculture.In this research we study on the basic of engineered phosphorous solubilizing microbeswhich isolated from four grape-producing areas in Northwest China. We engineered efficientindigenous grape rhizosphere soil phosphate solubilizing bacteria strains JYR-3, JYR-7,JYR-8and JYR-12double labeling of genetic transformation, such as expressing a greenfluorescent protein (GFP) and antibiotic resistance marker constitutively as a tracer means.We studied the colonization of young plants of V. vinifera L. cv. Cabernet Sauvignon bylabeled efficient phosphate solubilizing strains in the greenhouse pot experiment. We used theelectroporation method for the Gene transformation. Phosphate solubilizing bacteria JYR-3was labeled with contains pGFP4412and pGFP166which have different promoter-drivensgreen fluorescent protein gene, plasmid pGFP166was transferred to the target dissolvedphosphorus strain JYR-7and JYR-8, then we used resistance screening to determine theirrates of expression and observed by fluorescence detection in the Blu-ray irradiation and sawa clear emissive green fluorescence.With the help of fluorescence microscope and confocallaser scanning microscopy, the dynamic law of the survival characteristics of the targetmicroorganisms in grape rhizosphere colonization was studied. Conclusions are as follows:1. In stability test, the growth curve difference of the transformation strain and theoriginal strain is unconspicuous. It is stable to the transferred plasmid which has stabilizegenetic, compared with the original strain, the dissolution of phosphorus of marked strain,does not reduce, which to prove that the physiological and biochemical effects on the target strain which the plasmid was transferred to is unobvious, does not affect their normal growthand reproduction.2. We carry on the survival and colonization Position of the marked strain experimentresearch, Test results demonstrated that marked strains can survive in the grape rhizosphere.In the plate dilution gradient method for testing,its main root colonization converges in thecell gap of grape roots and the sunken part of the roots. labeled strains survive in the soilgreater than104cfu g-1in the soil within20days; after seed soaking a month, its existencecan also be detected in the colonization of soil; further study by laser scanning confocalmicroscope detection of colonization, they mainly colonize in cross-site of taproots rootslateral roots of grape roots, they also exist at two flanks of the root apical parts. Great amountof them in the grape primary roots could be observed.