Genetically Marking Of Pseudomonas Fluoresence And Biosafety Assessment Of Engineered Strain

It is the precondition for biocontrol effect that biocontrol microbe can colonize, survive and live well in the fields. The survival of recombinant microorganism is one of main factors in biosafety assessment of genetically modified organisms (GMO). In recent years, a few new report genes have been used as efficient methods in the study of microcosmos and macroscopy. Meanwhile, the new techniques and methods are being utilized widely for biosafety assessment of engineered strains.The vector pJBA28, which carried gfp gene, promoter PAl/04/03 and mini-Tn5 transposon, was modified and constructed to be a medium plasmid vector pGFP containing PPSOS promoter. Plasmid pJBA28 and pGFP were transformed into E. coli S17-1 respectively, and sequently the conjugation was carried on. S17-1 (pJBA28) and S17-1/ (pGFP) were recipient strains, and P303 was donor strain. The conjugants P303ml and P303m3 were acquired, which fluoresced steadily under 488nm. Both PCR and Southern blotting analysis demonstrated that gfp gene had been inserted into P303 chromosomal DNA randomly. SDS-PAGE assay indicated that the expression of GFP in P303m3 (carried PAl/04/03) Was weaker than in P303ml (carried Pp303), and the expression of gfp gene located in the chromosome was weaker than in the plasmid. The growth curve of P303ml certified that it had the same growth speed as the wild strain P303. Genetic stability research showed that the stability of gfp gene remained 100% after 96 hr. The engineered Pseudomonas fluorescens BioP8, which carried three Bt cry genes, was obtained by electroporation and it showed high insecticidal activities against larvae of Plutella xylostella in lab bioassay. BioP8 and P303ml remained strong antifungal activity as P303 against plant pathogenic fungi: Alternaria brassicae, Boreytis cinerea Pers., Phytophthora nicotianae, Rhizoctonia solani, Sclerotinia sclerotiorum, Penicillium and Colletotrichum orbiculare.The survival and colonization of P303, P303ml and genetically modified derivative BioP8 in the natural soil in the greenhouse, around the rhizosphere and the phyllosphere of cabbage in the field were studied by selective culture and PCR identified methods. The results showed that the total population of culturable bacteriain the soil and in the rhizosphere was around 10 CFU/g soil (wet weight) and 1010CFU/g roots (wet weight). The population of tested strains retained 104 ~106 CFU/g soil in natural soil within 40 days and no less than 10s CFU/g roots in rhizosphere of cabbage within 30 days. However, the total population of culturablebacteria on the phyllosphere of cabbage fluctuated between 10 and 10 CFU/ cm leaf, and that of P303 and BioP8 declined from105 to 103 and 102 CFU/ cm2 leaf from the first day to the 3rd day sharply, then increased and kept steadily between 103 and 104CFU/ cm2 leaf (5th-15th days). In conclusion, BioP8, P303ml and P303 hadsimilar dynamics of colonization and could survive no less than 60, 50, 15 days in the soil, rhizoshpere and phyllosphere respectively. No remnants and diffusion could be detected in the soil, in the rhizosphere or the phyllosphere of cabbage after one season (120 days). The engineered strain BioP8 was determined to be safe to the environment. It was also found that the ability of colonization of all tested strains in the soil and rhizosphere was much stronger than that in phyllosphere, and P303 was appreciably stronger than BioP8.An effective method of total DNA extraction of microorganism in soil and phyllosphere was established and the yield of total DNA in the soil sample was up to 10ug DNA/g soil (wet weight). The 16S rDNA V6 region of soil and phyllosphere bacteria were amplified, and the result of DGGE (Denaturing Gradient Gel Electrophoresis) showed ecological diversity.