Development Of RpoC Gene As A New Molecule Maker For Investigation Of Intestinal Flora And Its Application In Microecology Of Traditional Chinese Medicine

Objective:Intestinal microbiota can be regarded as a metabolic "organ" and participate in the life activities of the host.At present,16 S rRNA gene technology is the gold standard in the isolation,identification and classification of intestinal flora.However,16 S rRNA gene technology has limitations.This paper aims to develop new methods to supplement 16 S rRNA gene technology and analyze its application in traditional Chinese medicine microecology.Method:1.Based on perl language,the limitations of 16 S rRNA gene technology were analyzed theoretically,and the whole genome of typical intestinal bacteria was obtained from NCBI database.The fragments with high homology were found by comparison,and the universal primers were designed.The universal primers that can stably amplify were found by testing.2.The total DNA of intestinal microflora was artificially simulated,and the different primers of rpoC gene and 16 S rRNA gene were used to amplify them.The diversity characteristics were obtained based on the first generation Sanger sequencing.3.The total DNA of simulated intestinal microflora,normal rat intestinal microflora and normal human intestinal microflora were obtained,and the rpoC gene primers and 16 S rRNA gene primers were used to amplify them.Based on the second and third generation sequencing,the diversity characteristics were obtained and compared comprehensively.4.The total DNA of intestinal microflora in rats with AAD treated by Lizhong decoction was obtained,and the rpoC gene primers and 16 S rRNA gene primers were used to amplify the total DNA.The diversity characteristics were obtained based on the second and third generation sequencing,and the comprehensive comparison was carried out.Result:1.16 S identification of 11 typical intestinal microflora(Bacteroidetes,Firmicutes,Alphaproteobacteria,beta Proteobacteria,gamma Proteobacteria,deltaproteobacteria,epsilon Proteobacteria,Actinobacteria,planctomycetes,verrucomicrobia,spirochaetes)The average copy numbers of 16 S rRNA genes of Alphaproteobacteria,beta Proteobacteria,gamma Proteobacteria,Firmicutes and Bacteroidetes were 2.55,3.8,5.19,6.57 and 3.46,respectively.More than 70% of the strains had multiple copies of 16 S rRNA genes,and more than 8 copies of 16 S rRNA gene sequences were dissimilated.Among the 312 rpoC functional genes randomly extracted,the overall average sequence difference rate was concentrated in 20-60%,much higher than 5-30% of 16 S rRNA genes.Compared with the strains in the phylum,the nucleotide similarity of Proteobacteria phylum was 40-44%,and Firmicutes phylum was 42-45%;based on the comparison of protein level,the similarity of Proteobacteria phylum was low,only 23-30%,and Firmicutes phylum had 45-57% high similarity.There were 13 conserved regions which could be used to design primers and probes.Theoretically,multiple copies of 16 S rRNA genes are common,and the base difference rate of functional genes is higher than that of 16 S rRNA genes2.Based on the first generation Sanger sequencing,4 pairs of 16 S were compared Among them,Bacteroidetes accounted for 8.97%,Firmicutes accounted for 62.82%,Proteobacteria accounted for 28.21%;the diversity of primers 8F / 518R: Bacteroidetes accounted for 0.52%,Firmicutes accounted for 71.35%,Proteobacteria accounted for 28.36% The results of primer 338 F / 806 R showed that Bacteroidetes accounted for 5.38%,Firmicutes accounted for 61.83%,Proteobacteria accounted for 32.79%,genus level hit rate was 73.3%;primer 515 F / 926 R showed that Bacteroidetes accounted for 13.30%,Firmicutes accounted for 59.04%,Proteobacteria accounted for 28.36%,genus level hit rate was 80%;primer 27 F / 926 R showed that Bacteroidetes accounted for 13.30%,Firmicutes accounted for 59.04%,Proteobacteria accounted for 28.36%,genus level hit rate was 80% The results of 8F / 1492 R amplification showed that Bacteroidetes accounted for 0.50%,Firmicutes accounted for 60.50%,Proteobacteria accounted for 39.00%,and the genus level hit rate was 80%;the results of rpoC primer amplification showed that Bacteroidetes accounted for 36.68%,Firmicutes accounted for 35.69%,Proteobacteria accounted for 27.63%,and the genus level hit rate was 66.67%.Based on the gate level diversity survey,338 F / 806 R and 515 F / 926 R obtained closer diversity results,and rpoC preferred to amplify Bacteroidetes.Based on the diversity of genus level,the hit rate of four pairs of universal primers for 16 S rRNA genes were higher than that of rpoC genes,but they still could not amplify all genera,such as clostridioides and Salmonella,while rpoC genes could be amplified well.3.Based on high-throughput sequencing,16 S rRNA gene and rpoC gene were compared to describe the structure of simulated intestinal flora(M),normal rat intestinal flora(R1,R2)and normal human intestinal flora(F,Q)The results of rRNA high-throughput sequencing were as follows: Bacteroidetes accounted for 9.03%,Firmicutes accounted for 58.49%,Proteobacteria accounted for 32.48%.The results of rpoC based high-throughput sequencing were as follows: Bacteroidetes accounted for 18.60%,Firmicutes accounted for 57.24%,Proteobacteria accounted for 24.09%.Results: Based on rpoC gene molecular markers,115 OTUs were obtained from simulated intestinal flora(M),437 and 307 OTUs were obtained from normal rat intestinal flora(R1,R2)and 392 and 309 OTUs were obtained from normal human intestinal flora(F,Q)Twenty nine OTUs were obtained from simulated intestinal flora(M),628 and 552 OTUs from normal rat intestinal flora(R1,R2)and 148 and 165 OTUs from normal human intestinal flora(F,Q).On the rarefaction curve,16 S rRNA gene tended to be stable at 20000 reads,while rpoC gene curve did not.The results of OTU counting showed that rpoC gene had advantages in the number of OTU like m,F and Q,while 16 S rRNA gene had advantages in the number of OTU like R1 and R2.At genus level,neither 16 S rRNA gene nor rpoC gene could hit all genera.The hit rate of rpoC gene was lower than that of 16 S rRNA gene.4.Based on two kinds of molecular markers,we investigated the changes of intestinal microflora structure in rats with AAD treated with Lizhong Decoction(DML1: fecal sampling of normal fed rats;DML2: fecal sampling after one week of antibiotic gavage;DML3: fecal sampling after one week of Clostridium difficile gavage;DML4: fecal sampling after Lizhong Decoction treatment)The change trend of F / B ratio in AAD rats was the same during the treatment.At the genus level,the rpoC gene and 16 S rRNA gene hit 65 and 129 genera respectively,and 24 genera were hit by the two methods.The dominant genera obtained by the rpoC method were lactobacillus,Campylobacter,Bacteroides,Prevotella and barnesiella The results showed that the dominant genera of bacteria obtained by rRNA gene method were Bacteroides,Lactobacillus and Bacteroides;in DML2 samples after intragastric administration of antibiotics,rpoC gene and 16 S rRNA gene hit 11 and 67 genera respectively,and the two methods hit 8 genera together.The dominant genera obtained by rpoC method were Bacteroides,Escherichia and 16 S rRNA gene The results showed that the dominant genera obtained by rRNA gene method were Bacteroides,Escherichia and Bacteroides;in DML3,rpoC gene and 16 S rRNA gene hit 28 and 71 genera respectively,and the two methods hit 12 genera together.The dominant genera obtained by rpoC gene method were Bacteroides,lachnoclostridium,parabacteroides and 16 S rRNA gene The dominant genera obtained by rRNA gene method were Bacteroides,Bacteroides and ruminocaceae;after Lizhong decoction treatment,the rpoC gene and 16 S rRNA gene hit 63 and 110 genera respectively,and the two methods hit 30 genera together.The dominant genera obtained by rpoC gene method were barnesiella,mordavella,Francisella,Prevotella,16 S rRNA The dominant genera obtained by rRNA gene method were bacteroidales,Lactobacillus,Prevotella and Bacteroides.It is not difficult to see that both 16 S rRNA gene and rpoC gene can not obtain all genera at genus level.The hit rate of rpoC gene was lower than that of 16 S rRNA gene.The number of rpoC genes in both genera showed the same trend.Conclusion:(1)The results showed that nearly 70% of the 16 S rRNA genes had multiple copies,while rpoC gene was single copy The base difference rate of rRNA gene is 5-30%(DNA level),while that of rpoC gene is 20-60%,which has higher base mutation rate.Bioinformatics analysis of genome shows that rpoC gene is a potential molecular marker gene;(2)No matter for different environmental samples or using different sequencing techniques(the first,second and third generation sequencing techniques),16 S rRNA gene based microflora detection techniques can not fully obtain the true diversity of microflora,and other molecular markers(such as the complement and verification of rpoC gene molecular markers)are needed;(3)In this paper,rpoC gene is used as a molecular marker in the study of bacterial diversity in AAD animal model.The results are similar to 16 S rRNA gene technology.However,rpoC gene technology can make up for the shortcomings of 16 S rRNA gene technology in the detection of bacterial abundance and identification of related bacteria;(4)The advantage of high sequencing depth of the second and third generation sequencing technology can not make up for the inherent defect of 16 S rRNA gene,which is "relatively low resolution / unable to exhaust all the diversity",and rpoC gene molecular markers can play a complementary role in 16 S rRNA gene technology.