Dynamic Variation Of Environmental DNA And Acceptor Function In Bacterial Genetic Transformation

Evaluating the ecological influence of Genetic Modified Organisms (GMOs) after releasing to environments plays a key role in their development and utilization. The hazards coming from Horizontal Gene Transfer (HGT) in utilization of biological resources grow more and more interest. There are four kinds of mechanism of HGT found in laboratory: transformation, conjunction, transduction and cell fusion. Transformation is the acquisition of new genetic material by incorporation of added DNA, whose expression then changes the properties of the recipient cell. Transformation includes three factors: donor function, DNA in environment and host function. This study focuses on the latter two.Study on host function:1) DTA-TG studies show the DTA and TG curves of Escherichia coli competent cells induced by CaCl₂ have changed and the water content of competent cells increases, which indicate they are influenced either in quantity or in quality. That is to say the competent cells differ from the origin. This suggests the development of E.coli competence might be physiologically regulated.2) Mg²⁺ and Ce³⁺ substituted Ca²⁺, the main factor in competence development of E. coli, respectively. The results indicated that Mg²⁺ and Ce³⁺ could not induce the competence development and transformation of E. coli independently. When Mg²⁺ and Ce³⁺ were used in low concentration in transformation mixture respectively, the transformation efficiency increased, but when used at high concentration they inhibited the transformation process. These suggested that Ce³⁺might be involved in the physiological regulation in E. coli, as similar as Ca²⁺ was.3) Although EGTA is required for chelating bivalent cation in plasmid transformation system, Ca²⁺ can increase the transformation efficiency of Bacillus subtilis. We suggest that Ca²⁺ can restore the B. subtilis cells which are not iningredity because of EGTA. So it is propitious to B. subtilis natural genetic transformation. While Ce3+ decreases the transformation efficiency of B. subtilis by lowering the cell survivability due to its toxicity.4) All the results of B. subtilis transformation test with different methods testify that B. subtilis cell could turn to be competent in natural sand environment.5) We also study the function of melanin in transformation of B. subtilis, which indicates that melanin in low concentration not only stimulates cell growth but also increases the transformation efficiency. This should be taken into account when we develop and utilize melanin.Study on DNA in environment:1) This study is about the distribution and transformation activity of the DNA after it released into the sand. It shows that the DNA absorbed by sand preserves the transformation activity even after elution and extraction. This indicates that the DNA and sand form the complicated combination instead of the simple one.2) The distinct resistibility to DNase I and transformation ability indicate that the DNA and sand form at least three different kinds of complex.3) The transformability of adsorbed DNA by sand could be monitored over 35 days, while the stability of free plasmid without protection of sand only maintained for 7 days.4) In the presence of competent cells, the DNA with transformability, released from organism, prefers being accepted by competent cells to being absorbed by sand directly. The results also suggest that the transformability of sand adsorbed DNA correlated with form of the DNA and accessibility to competent cells, but concentration of DNA seems unimportant.These results provide lots of valuable information about the storage of DNA in environment as well as the interaction, horizontal gene transfer and ecological effect between the DNA and microorganism around it.