This experiment was carried out to establish an effective method for detecting the impact of virus resistant transgenic wheat on its environment bacteria. Variable region fragments of 16S rDNA V3 were amplified by specific and sensitive primers of bacteria, analyzed by denaturing gradient gel electrophoresis (DGGE), and applied to evaluate the effect of transgenic wheat on the soil bacterium, according to the change of electrophoretic bands. The results showed that the DNA by this extraction method could be used in PCR without purification. On DGGE profiles, the lighter bands of each lane represented their corresponding dominant microorganisms. The influence of transgenic wheat and non-transgenic wheat on soil microbes by the number and brightness of the bands were obtained, so microorganism population fluctuation could be demonstrated effectively with the method.

土壤微生物的多样性是保持农业生态系统稳定的基础,而作物的改变对土壤微生物的多样性结构具有显著的影响。转基因作物被引入到农田后所带来的微生物群落变化及对农业生态系统所带来的不确定因素,已成为研究热点。本文着重介绍了当前土壤微生物多样性研究中常用的3种基于16S rDNA的技术:变性梯度凝胶电泳(DGGE)、扩增性核糖体DNA限制酶切片段分析(ARDRA)和末端限制性酶切片段长度多态性(T-RFLP)技术,分析比较了各种技术的原理、优点及其应用局限性,并简短综述了这些技术近年来的应用。

The VH and VL gene fragments were first amplified from cDNA of hybridoma cell line specific to deoxynivalenol,integrated as ScFv with a pair of linker primers,cloned into phagemid vector pCANTAB-5E,and then transformed into Escherichia coli TG1.To produce phage-display ScFv,the helper phage M13KO7 was added to the cultures when bacteria were grown up to the log-phase,in which ScFv antibody was displayed in N-terminus of the bacteriophage coat protein pⅢ.The phage antibody contained 1.1×105 independent clones,and the titer of which was about 1.3×108 pfu.Enzymatic digestions of 10 random clones showed that the recombinant plasmids contained an intact ScFv gene.These results indicated that the phage antibody library was constructed successfully.