Molecular Mechanism Study On The Differences Of Chemical Constituents In Coptis Chinensis Franch.from Different Producing Areas Based On Transcriptome Sequencing

Coptis chinensis Franch.is a medicinal plant of the genus ranunculaceae,whose main medicinal parts are rhizomes.It mainly contains many alkaloids such as berberine,coptisine,palmatine,and epiberberine,among which the concentration of berberine is the highest.The expression levels of functional genes in different plants of C.chinensis were different,which caused the difference in the content of secondary metabolites.However,the quality of medicinal materials was not easy to control due to its many production areas,which led to the uneven quality of medicinal materials in the market.In this study,six samples of C.chinensis from different producing areas were selected as the research objects.High performance liquid chromatography(HPLC)was used to detect six alkaloids content,and three producing areas(Zhenping Shaanxi,Zunyi Guizhou and Shizhu Chongqing)with different content were selected.The samples of C.chinensis were used for transcriptome sequencing analysis;transcriptome sequencing was performed on three selected samples from Illumina HiSeq 2500,three biological replicates were performed for each sample,and the results were selected for the corresponding genes and sieved differences.Functional annotation and analysis of the gene,and finally the relevant candidate genes for the berberine synthesis pathway were unearthed,and the expression level of the candidate gene was verified by qRT-PCR consistent with the transcriptome sequencing results.The main findings are as follows:(1)HPLC analysis of the content of alkaloids in C.chinensis.The results showed that there were six types of alkaloids in the samples of C.chinensis in the six producing areas,but the content of each alkaloid was quite different.The total alkaloid content in the six producing areas was ranked as follows: Shizhu Chongqing > Lichuan Hubei>Zunyi Guizhou> Beichuan Sichuan > Longshan Hunan≥ Zhenping Shaanxi,of which the highest content of alkaloids in Shizhu Chongqing,indicating that the quality of medicinal materials in Shizhu Chongqing is the best.According to the results of the assay,samples from Shizhu Chongqing,Zunyi Guizhou and Zhenping shaanxi were screened for transcriptome sequencing;(2)The transcriptome of C.chinensis of Zhenping Shaanxi(T01),Zunyi Guizhou(T02)and Shizhu Chongqing(T03)was selected to obtain 14.89 Gb clean datas,and the percentage of base Q30 was 90.41% or above;The three origins of T01,T02 and T03 were sequenced to obtain 25088487,23905895,and 24723011 Clean reads respectively.The number of mapped reads obtained by comparison was25088487,23905895,and 24723011,respectively;(3)After sequencing the T01,T02 and T03 samples,a total of 9441697 contigs were obtained after software assembly and splicing.After further assembly,155710 transcripts were obtained,and 56671 pieces of redundant alternative splicing transcripts were removed.Unigenes has a N50 of 858 bp and an average length of about 492 bp.Use GetORF software forecast unigenes of CDS,CDS received a total of up to 2754420656028 bases;(4)Functional annotation of unigenes of C.chinensis,in which COG database received comments of 14035,GO database 24991,KEGG database 9462,Swiss-Prot database 21742,NR database 36935;a total of 24991 unigenes were annotated to128452.In the GO database entries,the unigene numbers of the three GO classifications of molecular function(MF,Molecular Function),cell composition(CC,Cellular Components)and biological process(BP,Biological Processes)are 38880,30102,59470,respectively;a total of 19274 unigenes is classified into the COG database;(5)MISA performed SSR analysis,and 6224 SSRs were identified in 14585 unigenes with a length of 1kb,among which the number of single bases was the highest;SOAPsnp analysis showed that 1037576 SNPs were identified,of which970763 is a biallelic;(6)After the transcriptome sequencing data of T01,T02 and T03 were compared and mapped to 155710 transcripts,the number of mapped reads was25088487,23905895 and 24723201,respectively.Articles,accounting for 81.69%,83.24%,and 80.29% of their respective clean reads;(7)The number of differentially expressed genes in C.chinensis from different habitats was 6,928 between Zhenping Shaanxi and Zunyi Guizhou(T01vsT02),7068 between Zhenping Shaanxi and Shizhu Chongqing(T03vsT01),7939 between Zunyi Guizhou and Shizhu Chongqing(T03vsT02).KEGG’s annotations were classified according to the type of pathway in KEGG.The results showed that 11573 unigenes recognized the 129 KEGG pathways,and the most abundant pathway was "ribosomes" with a total of 948 unigenes,screening for candidates involved in the cyclaline quinoline biosynthesis pathway.Gene,a total of 32 unigenes involved in the biosynthetic pathway of isoquinoline alkaloids;(8)Validation of candidate genes for C.chinensis in the isoquinoline alkaloid biosynthesis pathway: c101794,c168605,c175479,c177134,c150791,c156287,c162923,c129387,c165925 and c174243,qRT-PCR results and transcriptome sequencing results are consistent;In summary,this study used HPLC to detect the content of C.chinensis in different habitats,and screened samples with large differences in content for transcriptome sequencing,obtained a large number of its gene information,and related gene annotation and significantness of differential genes by bioinformatics methods.The experimental results analyzed the molecular mechanism of the formation of C.chinensis in different habitats;By mining the relevant candidate genes of berberine synthesis pathway,combined with real-time PCR to verify these candidate genes of C.chinensis,the experimental results was provided for gene regulation and genetic transformation of the berberine synthetic pathway with datas.