Analysis Of Diazotroph Communities In Transgenic Rice Rhizosphere

The environmental safety issues of transgenic rice are the focus of attention of researchers . At present ,the impact of transgenic rice on soil bacterial communities still little is known . One crucial function carried out by soil microorganisms is nitrogen fixation, which is the major source of nitrogen for many natural ecosystems. It is important primarily because nitrogen often is the limiting nutrient in many terrestrial ecosystems . In this paper, we take advantage of the method of conserved genes to evaluate the diversity of diazotroph communities in transgenic rice rhizosphere. The major study content and conclusion shows as follow:1,Comparison of three extraction methods of soil microbial DNA.Three commonly used methods for soil microbial DNA extraction including Martin method, improved high-salt concentration method and the kit method were assessed in this paper. The evaluation was based on the yield and purity of extracted total DNA, the amplification of 16S rDNA V3 variable region and the subsequent analysis of amplified products by DGGE (denaturing gradient gel electrophoresis). The results showed that each of three methods of DNA extraction could meet the requirements of analysis of soil microbial diversity.The DGGE strip of kit method was more homogeneous , could represent the soil microbial diversity. Compared with the kit method , because of the purification of Martin method and improved high-salt concentration method were not enough to improve the law completely ,the impurities in DNA extraction may affect the PCR amplification reaction,and PCR amplification products had the small number of band changes of DGGE analysis , cause a devitation of analysis of soil microbial diversity . To ensure the reliability of the analysis of soil microbial diversity , the ideal solution was DGGE combinate the recycle of purpose of strip and sequence.The kit method was simple, the quality of extracted DNA was higher, but the yield of DNA was lower and the cost was more expensive. Martin method and improved high-salt concentration method both got higher yields of DNA with lower cost than the kit method but spent longer time. The purity of DNA were lower than one extracted by the kit method, but it would not affect the following PCR and DGGE analysis significantly. In this paper , kit method was chosen as the method that extract soil DNA in the rhizosphere of rice . 2,The impact of transgenic rice on the rhizosphere diazotroph community.The trial consisted of untransformed controls-zhongjian , transgenic rice-kefeng 6 (transformed with the Bt gene),and kangyou 97 (transformed with the Xa21 gene) .Diazotroph diversity was assessed by extracting soil genomic DNA and amplifying a region of the nitrogenase reductase (nifH) gene, followed by cloning and sequencing. we calculated richness estimators (bias-corrected Chao1 and bootstrap values)and diversity indices (Shannon and Simpson) , and the data there was no differernt significantly. We performed a dendrogram analysis using the nifH OTU sequences and the nifH sequences from known organisms to infer the possible affiliations of our different OTUs , and found that the majority of clones were related to two groups : the classes of proteobacteria indudingα-Proteobacteri ,γ-Proteobacteria ,β-Proteobacteria andδ-proteobacteria .The other guoup is Firmicutes .More than half of nitrogen fixation in the rhizosphere of zhong jian and kefeng 6 belong to Gammaproterbacteria ,the nitrogen fixation in the rhizosphere of kangyou 97 was lower in this bacteria class . There were no differences significantly among three rice in the other bacteria class . The vast majority of nitrogen fixation were clustering within the group I nitrogenase , and the group I nitrogenases were typical Mo-Fe nitrogenases , whereas a small number of them belonged to group II nitrogenases that are anaerobic Mo-Fe nitrogenases .Analysis revealed that nitrogen fixation communities did not differ significantly among the untransformed control, transgenic rice-kefeng 6 (transformed with the Bt gene) , and kangyou 97 (transformed with the Xa21 gene) . We therefore conclude, in the absence of evidence that the transgenic rice had an effect on diazotroph communities.