Study On Marking Of Pseudomonas Fluorescens By Red Fluorescent Protein Gene

Genetically modified microorganism (GMM) plays a significantly important role in agricultural production. Its potential biosafety to the natural environment is being concerned very much. It is molecular ecology technology that offers effective means and technical support for verification of bacterial ecological behavior. Pseudomonas fluorescens is main biocontrol bacterium, effective ecological studies of it, especially engineered are more and more enhanced by the use of genetically marked isolates to facilitate monitoring. Therefore it is very important to construct the eminent mark system for genetically engineered Pseudomonas fluorescens strains.In this study, Pesticidal activity of BioP8 was lower than chemical pesticide against Plutella xylostella control in the fields. It was gradually enhanced for BioP8 to control Plutella xylostella after seven days. The biocontrol result was 46.4% after seven days, 85.7% after 15 days, and excelled chemical pesticide. The number of main natural enemies of field controlled by BioP8 was almost equal to the negative control area while that by chemical pesticide was dramatically decreased 68.7%, 62.5%, 36.7% after 3 days, 7 days, 15 days, separately. Different treatment had different influence on the population of arthropod in the cabbage field. The results of investigation elucidated that there was difference in abundant degree of different arthropod species. Abundant degree of pest species was in turn of negative control field > pesticide treatment field > BioP8 treatment field. The abundant degree of natural enemy species also showed different, it was in turn of BioP8 treatment field > negative control field > pesticide treatment field. The results indicated that the chemical pesticide had a great influence on natural enemies and spiders, the quantity of pests kept a lower level in the treatment field, the spider and natural enemy kept higher in quantity. Evaluation result of engineered strain BioP8 to growth and development of cabbage indicated that there was no negative influence against crop, and no cabbage pathogenic diseases could be induced in the whole season. The cabbage grew well in the field treated with BioP8, as well as with pesticide, while obviously was better than the negative CK.The result of RFP dealt with insect digestive juice indicated RFP could resist proteinase among midgut digestive fluid. RFP was very steady in SDS-polyacrylamide gel, and emitted the normal red fluorescence. All the assayresults showed that RFP was stable, and these characteristic could be applied on the study of action mechanism between Cry protein and receptors of insect pests.expression cassette carrying red fluorescent protein (RFP) gene was constructed to generate pJH3. The new vectors pJH4 and pJH5 were obtained by insertion of rfp gene into mini-Tn5 and shuttle vector pSPCPa-lac respectively. Then rfp gene was transposed and inserted into chromosome of Pseudomonas fluorescens P303 and the engineered bacterium BioP8, getting modified strains 8'P303 and 8'BioP8 separately. pJH5 was transformed into P303 to generate 12P303. The engineered bacteria using plasmid marker emitted bright red fluorescence under 366 nm UV light, while engineered bacteria of chromosome marker emitted a slightly weak red fluorescence under daylight,but brighter under UV light than that under the daylight. All bright red fluorescence elicited from red fluorescent protein produced by the engineered bacteria could be detected very well by confocal laser scanning microscopy (CSLM) readily and there weren't significant difference. When the images were converted into three dimension, it became obvious that the intensity of fluorescence produced by plasmid marker was much brighter that by chromosome marker. The results obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 12P303 showed that yield of RFP increased appreciably up to 32 h, and then there was no further increase. In contrast, the 8'P303 yield of RFP had no appreciable increase from 12 h to 60 h. The growth curve of 8'P303, 12P303, P303 certified that it had no significant different at growth speed. Genetic stability assay result of a couple of recombinants showed it remained above 98% after 96 h. 8'P3O3 and 12P303 remained strong anti-fungi activity as P303 and had no significant different. The survival and colonization of 8'P303, 12P303 and P3O3 in the soil collected from a farm in green house were performed by selective culture. The results indicated that the total population of culturable bacteria in the soil was around 108 CFU/g (wet weight of soil). The population of tested strains showed the trend of gradual decline in thirty days. P303 and the engineered bacteria had similar dynamics of colonization and could survive no less than 30 days in the soil. The result of biosafety research of the recombinant bacterium BioP8 demonstrated that total population of culturable bacteria on the leaves of cabbage was around 108CFU/cm2; the population of tested engineered strains retained 103 CFU/g; the total population of culturable bacteria in the soil was around 108 CFU/g; the population of tested engineered strains retained 102 CFU/g. Bascillus had similar dynamics of colonization and was about 106 CFU/g before and after spraying BioP8. After 30 days, the total population of culturable bacteria was about 108 CFU/g. No Rif/Km/Spc resistant colonies were found in the soil out of five meter...