Optimization Of Soil RNA Extraction Conditions And Their Applications In Bacterial Diversity Study

The extraction of total RNA from soil microbes is the basis for studying the molecular ecology of soil microbes,in studying the relationship between the distribution and diversity of soil microbial community function,you need to extract high quality RNA samples from the soil.RNA research at the transcriptional level,and is mainly based on physiological and biochemical processes of microorganisms,research in the RNA level,the ability to respond in time to discover when changes occur to microbial external environment.Therefore,establish a method that total RNA extraction from soil can provide the basic material for the study of microbial communities and functionality.This study establish a method that total RNA extraction from soil,which the method can be a stable and efficient extraction soil Total RNA.Loam was used as the research object,using the method repeated six times,the extraction result show:loam RNA concentration average of 167.15 ng/?L,coefficient of variation of only 10.29%.Compared with the Omega kit,the extraction efficiency was higher than kit.The efficiency of clay extraction was four times that kit,and the efficiency of sand extraction was not significantly different from kit.In addition,the soil microbial community diversity and abundance of three soil DNA and cDNA samples were studied using Illumina MiSeq second generation high throughput sequencing technique,and compared the diversity of autotrophic carbon-fixing microbial community diversity at the DNA and cDNA level.The results of this research are as follows:1.The total RNA was extracted from three samples of loam,pure colony(Escherichia coli)and plant(Arabidopsis thaliana)by traditional Trizol method,the results showed that the Trizol method was poor in the extraction of total RNA from soil microbes,while the extract of E.coli and Arabidopsis thaliana was well,indicating that Trizol reagent is not suitable for soil samples with complex environments,presumabling that related to the heterogeneity of the soil microbial often mixed with soil particles and other impurities together,dispersed and wrapped microorganism relative soil.In addition,the Trizol reagent cleaves less than other lysing agents.2.On the basis of CTAB-SDS method from the extract by changing the pH,precipitation time and cracking time,resulting in the RNA extraction method suitable loam,clay and sand,by contrast effect while Omega Company found soil RNA kit,kit Omega Company there are differences in the three soils applicability,applicability:sand>clay>loam,the quality difference CTAB-SDS extract optimization method on soil was not significant,RNA extracted from CTAB-SDS was extracted by DNA,RNA purification and cDNA synthesis,and could be used for the amplification of 16S and cbbM genes.3.The community structure of cbbM gene in loam,clay and sand was analyzed by Illumina MiSeq high throughput sequencing technique.The diversity of microbial community diversity of DNA and cDNA samples in three soils was compared.Results showed that the extraction of CTAB-SDS soil RNA optimization method can be applied to high-throughput sequencing analysis,in the diversity index,the soil samples of loam,clay and sand were higher than those of three soil samples,by comparing the DNA and cDNA samples,it was found that Acidithiobacillus,Sulfuritalea,Thiobacillus,Magnetospirillum,Thiohalomonas,Thioflavicoccus,Thiomonas,Acidihalobacter,Sulfuritalea,Ferriphaselus,Gallionella and Thiocystis were more obvious.Furthermore,the reproducibility of loam DNA samples was 94.98%,the reproducibility of sand samples was the worst,and the average similarity was only 49.74%.Between the overlapping stability,samples of clay loam and coefficient of variation was below 2%,the lowest loam DNA sample,while the worst sand sample stability.