Typical zonal soil such as Brown soil was sampled from Tianwai village,Taishan,Shandong Province in China.Two soil colloidal components i.e,fine clay (＜0.2 m) and coarse clay (0.2-2.0 m) were separated by centrifugation.Two treatments applied to fine and coarse clays were organic matter left on the samples (organic clays) and organic matter removed from the samples by H2O2 (inorganic clays).Brown soil was divided into four types of clays:coarse organic clay (0.2-2.0 μm,organo-mineral complexes),coarse inorganic clay (0.2-2.0 μm,H2O2-treated clay),fine organic clay (＜0.2 μm,organo-mineral complexes) and fine inorganic clay (＜0.2 μm,H2O2-treated clay).The adsorption,desorption and binding mechanism of DNA on Brown soil colloid or mineral such as montmorillonite,hydroxyaluminum-montmorillonite,kaolinite and goethite,the ability of transforming competent cells of bound DNA and the resistance to DNaseⅠ degradation,the PCR amplification of bound plasmid DNA and the effects of soil active particles on microbial metabolic activities were investigated.The main results were as following:1.Adsorption-desorptionof DNA,ATR/FTIR,circular dichroism (CD),fluorescence spectroscopy,microcalorimetry were used to clarify the adsorption mechanism of DNA on permanent-charge soil active particles.The maximum amount of DNA adsorbed followed the order:montmorillonitefine inorganic clay>fine organic clay>kaolinite>coarse inorganic clay>coarse organic clay.A marked decrease in the adsorption of DNA on organic clays and montmorillonite was observed with the increase of pH from 2.0 to 5.0.Little DNA was adsorbed by organic clays above pH 5.0.As for inorganic clays and kaolinite,a slow decrease in DNA adsorption was found with increasing pH from 2.0 to 9.0.Magnesium ion was more efficient than sodium ion in promoting DNA adsorption on soil colloids and minerals.DNA molecules adsorbed on soil colloids and minerals were desorbed by sequential washing with 10 mmol/L Tris-HCl,100 mmol/L NaCl and 100 mmol/L sodium phosphate buffer at pH 7.0.A percentage of 53.7%~64.4% of DNA adsorbed on organic clays and montmorillonite was released,while only 10.7%~15.2% of DNA on inorganic clays and kaolinite was desorbed by Tris-HCl and NaCl.The percent desorption of DNA from inorganic clays,organic clays,montmorillonite and kaolinite by sodium phosphate buffer was 39.7%~42.2%,23.6%~28.8%,29.7% and 11.4%~29.7%,respectively.DNA adsorption on organic clays was endothermic (1.1 ＜△Hads＜3.5 kJ/g),whereas that on inorganic clays was exothermic (-0.3kJ/g＜△Hads＜-0.1 kJ/g).Dehydration effects and electrostatic interactions dominated DNA adsorption on organic clays and montmorillonite,and DNA was adsorbed predominantly by ligand exchange and possibly hydrogen bonding on inorganic clays and kaolinite.ATR/FTIR spectra showed that the binding of DNA on kaolinite and inorganic clays changed its conformation from the B-form to the Z-form,whereas montmorillonite and organic clays retained the original B-form of DNA.A structural change from B- to C-form in DNA molecules desorbed from kaolinite was observed by CD spectroscopy and confirmed by fluorescence spectroscopy and DNA molecules desorbed from soil colloid or montmorillonite were still B-form.2.The effects of various organic and inorganic ligands on DNA adsorption on active soil particles were studied.An obvious decrease in DNA adsorption was observed on montmorillonite and kaolinite with increasing anion concentrations from 0 to 5 mmol/L.However,the amount of DNA adsorbed by montmorillonite and kaolinite was enhanced when ligand concentration was higher than 5 mmol/L.In the system of soil colloids,with the increase of anion concentrations,a steady decrease was found and the ability of ligands to depress DNA adsorption followed the order of phosphate>citrate>tartrate.Compared to inorganic clays,a sharp decrease in DNA adsorption was observed on organic clays with the increase of ligand concentration.The results indicated that the influence of anions on DNA adsorption varies with the type and concentration of anion as well as the surface properties of soil components.Introducing DNA into the system before the addition of ligands had the maximum amount of DNA adsorption on soil colloids.Organic and inorganic ligands promoted DNA adsorption on montmorillonite and kaolinite when ligands were introduced into the system before the addition of DNA.3.Adsorption and desorption of DNA on Bacillus thuringiensis and Pseudomonas putida and their composites with soil colloids or minerals were investigated.B.thuringiensis and P.putida did not show significant difference in the amount of DNA adsorption although the two bacterial cells had different surface properties.DNA was adsorbed on bacteria mainly through van der Waals force and electrostatic force.Compared with B.thuringiensis,DNA adsorbed by P.putida was desorbed more easily.There was no significant difference in the amount of DNA adsorption on kaolinite between the absence and the presence of P.putida.Except for the effect of P.putida on DNA adsorption on kaolinite,the presence of B.thuringiensis and P.putida significantly promoted DNA adsorption on soil colloids and minerals,and the promotion of B.thuringiensis was stronger than that of P.putida in the system of soil colloids. 4.Adsorption of DNA on different hydroxyaluminum-montmorillonite complexes (Al(OH)x-M) containing 2.5,10.0 and 20.0 mmol coated Al/g clay (AM2.5,AM10 and AM20) was studied in Tris-HCl and sodium phosphate buffers at pH 7.0.The coatings of montmorillonite by hydroxyaluminum species decreased the amount of DNA adsorption,but increased the affinity of DNA adsorption.At the same pH,the amount of DNA adsorption on montmorillonite or Al(OH)x-M complexes in sodium phosphate was greater than that in Tris-HCl,suggesting that the nature of a buffer solution strongly affected DNA adsorption on clays.As for Al(OH)x-M complexes,the higher the level of Al(OH)x coatings,the lesser the amount of DNA was adsorbed in sodium phosphate buffer.The reduction of DNA adsorption in sodium phosphate buffer with the increase of the level of Al(OH)x coatings may be ascribed to the strong competition of phosphate anions with DNA molecules on surface sites of Al(OH)x-M complexes.An increase of the concentration of Ca2+ and/or a decrease of the values of pH helped DNA adsorption on montmorillonite and Al(OH)x-M complexes.The desorption percent of DNA from montmorillonite,AM2.5,AM10 and AM20 was 65.01%,30.00%,8.04% and 5.18% by Tris-HCl buffer.It suggests that the larger the OH-Al loading on M surface,the greater the binding energy of DNA.DNA adsorption on montmorillonite was endothermic (△Hads=1.15 J/g),whereas that on Al(OH)x-M complexes was exothermic (-9.50 J/g＜△Hads＜-6.64 J/g).The bases and phosphate groups of DNA are involved in DNA adsorption on clays and DNA changes its conformation from the B-form to the Z-form as the result of its binding on AM10 and AM20.Electrostatic forces,hydrogen bonding and ligand exchange dominated DNA adsorption on Al(OH)x-M complexes.SEM showed that a thin layer was formed on the surface of AM10 after the binding of DNA,while that was not observed on montmorillonite surface.5.Electrophoresis and thermometric TAM III were used to investigate the degradation of chromosomal DNA in the system of active soil particles or bound on soil colloid or mineral by DNaseⅠ.When nuclease concentration was 2.0μg/mL,DNA was completely degraded.In systems of kaolinite and coarse and fine inorganic clays,DNA was degraded to 2-4 kb segments at 20 μg/mL of nuclease.For DNA in systems of montmorillonite and coarse and fine organic clays,no evident change was observed in the patterns with 40 μg/mL nuclease in comparison with no nuclease.The heat released from the hydrolysis of DNA,free or bound on coarse inorganic clay,coarse organic clay,kaolinite and montmorillonite by the nuclease was -4.76,-4.06,-2.38,-2.36,-0.22 mJ,respectively.These results indicated that soil colloids and minerals could exert an effective protection for DNA to resist degradation by the nuclease.Among the soil colloids and minerals studied,montmorillonite and organic clays provide more protection for DNA against degradation by DNaseⅠ than kaolinite and inorganic clays.The protection of DNA was not a result of the adsorption affinity of DNA for soil colloid or mineral and the changes in DNA structure.The presence of organic matter and an efficient adsorption of nucleases on soil colloids and minerals appeared to be responsible for the lower degradation of DNA in soil ecosystems.6.The ability of bound plasmid p34S DNA on soil colloids and minerals to transform competent cells of CaCl2-treated Escherichia coli,and the resistance of bound plasmid DNA to degradation by DNaseⅠ were investigated.The transformation efficiency of bound plasmid DNA increased with increasing concentrations of Ca2+ at which soil colloid or clay mineral-plasmid DNA complexes were formed.Plasmid DNA bound by kaolinite showed the lowest transformation efficiency,and especially no transformants were observed with kaolinite-plasmid DNA complex prepared at 5-100 mmol/L Ca2+.Compared with organic clays and fine clays,plasmid DNA bound on inorganic clays and coarse clays showed a lower capacity to transform E.coli at different Ca2+ concentrations.Transformation by 10 μg of free plasmid DNA was inhibited 99.8% by 10 ng of DNaseⅠ.As for the same amount of plasmid DNA bound by soil colloids and montmorillonite,100 ng of DNaseⅠ resulted in 92.3%-93.8% inhibition of transformation by plasmid DNA bound on inorganic clays,whereas 2000 ng of DNaseⅠ caused only 64.0%-98.0% inhibition of transformation by that on organic clays and montmorillonite.The percentage of reduction of transformants by plasmid DNA bound on coarse clays was higher than that on fine clays.Montmorillonite,organic clays and fine clays showed stronger protective effects for plasmid DNA than that of inorganic clays and coarse clays.The adsorption affinity of plasmid DNA for soil colloid or mineral and a conformational change in the plasmid DNA molecule bound on clays may determine the efficiencies of transformation.7.The polymerase chain reaction (PCR) was used to amplify a 600-base pair (bp) sequence of plasmid pGEX-2T DNA bound on soil colloidal particles and three different minerals (goethite,kaolinite,montmorillonite).DNA bound on soil colloids,kaolinite,and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted 10- and 20-fold.The montmorillonite-DNA complex required at least 100-fold dilution before amplification was detectable.DNA bound on goethite was amplified whether the complex was used directly,or diluted 10- and 20-fold.The PCR amplification of mineral-bound plasmid DNA was markedly influenced by the types and concentrations of minerals used.8.The thermodynamic data of the metabolic activity of E.coli as influenced by soil colloids and minerals were analyzed.The growth rate constant (k) of E.coli in LB was 0.074/min,and the k values of E.coli in the system of coarse inorganic clay,kaolinite,coarse organic clay,montmorillonite and goethite were 0.073,0.058,0.054,0.045 and 0.020/min,respectively.It suggested that the selected soil colloids and minerals inhibited significantly the exponential growth of E.coli.The inhibitory ability of the three minerals on metabolic activity of E.coli followed the sequence of goethite>montmorillonite>kaolinite.Compared with inorganic clay,organic clay showed a higher inhibitory effect on metabolic activity of E.coli.