| Isoquinoline alkaloids(IQAs)are a large group of natural products.They are important pharmaceuticals that in Papaveraceae,Ranunculaceae,Magnoliaceae,Berberidaceae and Aristolochiaceae plants.Such as berberine has the effect that antibacterial,anti-inflammatory,antiviral,anti hyperglycemic and so on.The sanguinarine is a good antibacterial agent;papaverine is a muscle relaxant;morphine and codeine are good painkillers.Isoquinoline alkaloids biosynthesis starting from amino acid,such as berberine,sanguinarine,columbamine,plamatine and magnoflorine are starting by tyrosine with various enzymes.In addition,transcription factors are also a key regulatory gene for the biosynthesis of isoquinoline alkaloids,like the Cj WRKY1,Cjb HLH1,Ecb HLH1-1 and Ecb HLH1-2.At present,the main research target of isoquinoline alkaloids is Opium poppy,but the biosynthesis pathway of isoquinoline alkaloids in Ranunculus is less.Coptis chinensis Franch and C.teeta Wall belong to Ranunculaceae,is also an important medicinal plant resources in China,which contains a variety of isoquinoline alkaloids,including berberine,palmatine,columbamine and magnoflorine biosynthesis path known,while coptisine,epiberberine and jatrorrhizine is not clear,two kinds of Coptis are excellent materials for studying the isoquinoline alkaloid biosynthesis pathway and Coptis species differences.In this study,we used Illumina paired-end sequencing technology on the Illumina Hi SeqTM 2000 platform to generate a large-scale transcriptome data for the Coptis chinensis Franch and C.teeta Wall.Functional annotations were performed by sequence comparison with multiple public databases,and comparative transcriptomic analysis of the two Coptis species were also performed.In all,a set of putative unigenes were predicted to be involved in isoquinoline alkaloids biosynthesis.This research will provide important information for quality improvement of two kinds of Coptis,and will also facilitates the study of the biosynthesis of active ingredients.The main results are as follows:1.HPLC for two tissues of Coptis chinensis Franch and C.teeta Wall,we measure the berberine,coptisine,plamatine,epiberberine and jatrorrhizine content and difference.The content of alkaloid in the leaves of two kinds of Coptis were lower than that of root,and the content of coptisine,palmatine and berberine in Coptis chinensis root were significantly higher than that of C.teeta.2.To investigate the root transcriptome of Coptis chinensis Franch and C.teeta Wall,Illumina Hi Seq? 2000 sequencing platform was employed.A total of 84,199,094 clean reads were obtained from the c DNA library of C.teeta.And a total of 95,053,646 clean reads were obtained from the c DNA library of C.chinensis.All the clean reads were assembled de novo using Trinity program,the clean reads from C.teeta were assembled into 49,741 unigenes with an N50 of 1,310 nt,the clean reads from C.chinensis were assembled into 55,903 unigenes with an N50 of 1,306 nt.The unigenes were aligned to the four public protein databases including Nr,Swiss-Prot,COG and KEGG.In C.teeta transcriptome databas,57.20% unigene were annotated in the public databases.In C.chinensis transcriptome database,53.33% unigenes were annotated.3.Most of the enzymes involved in the biosynthesis of isoquinoline alkaloids were found in the transcriptome database of C.teeta and C.chinensis.Through data analysis,78 unigenes were found in C.teeta and 87 unigenes were found in C.chinensis to be the key enzyme in the biosynthesis.Among them,the 78 unigenes in C.teeta are all the key enzyme candidate genes in the known path,while the other 86 in C.chinensis are known,and the one is the key enzyme candidate gene in the putative pathway.By BLAST,We further screened out Ct0017663,Cc0020654,Ct0000676 and Ct0033162 as candidate genes for CFS and SPS,which the biosynthetic pathway of the epiberberine and coptisine we speculated.In addition,the biosynthetic pathway of two isoquinoline alkaloids were unknown,in this experiment,we according to the known path synthesis path inferred coptisine and epiberberine and screened related enzymes candidate gene from transcriptome data,further improve the relevant information of isoquinoline alkaloid biosynthesis.4.The content of alkaloid in the leaves of two kinds of Coptis were lower than that of root.Through the comparative transcriptome analysis,we observed the difference in the expression of related enzyme genes found in two kinds of coptis in leaves and roots.The result found the expression that roots were higher than leaves in C.teeta and C.chinensis.The difference of the content is explained from the molecular level,and it is indicated that the key enzyme candidate genes obtained in this experiment have certain accuracy.5.The transcription factor maybe regulate the isoquinoline alkaloid biosynthesis.In this experiment,Cj WRKY1,Cjb HLH1,Ecb HLH1-1 and Ecb HLH1-2 transcription factors homologous gene were obtained.Through differential expression and cluster analysis,the candidate genes of Ct0005642 transcription factor Cj WRKY1 in Coptis chinensis Franch were identified,and the Cc0023994 gene was the candidate gene of Cjb HLH1.Two kinds of Coptis chinensis were not found to be highly homologous to Ecb HLH1-1 and Ecb HLH1-2 transcription factors.6.In this study,based on the analysis of the SSR loci information in the transcriptomes of two kinds of Coptis chinensis which were Coptis chinensis Franch.and C.teeta Wall.,the SSR primers were designed to provide help for the development of new SSR markers.By using MISA,we screened all of 55 903 and 49 741 unigenes which were obtained from sequencing of transcriptomes of Coptis chinensis Franch.and C.teeta Wall.,and then analyzed the SSR loci information.SSR primers were designed by Primer 3.Furthermore,60 pairs of primers were randomly selected for the polymorphism analysis on 40 Coptidis(20 of each kind of Coptidis)plants.A total of 4,071 and 4,041 SSRs were found in the each transcriptomes,respectively.Di-nucleotide repeat and Tri-nucleotide repeat were the main type,account for as much as 87.71% and 87.58% of all SSRs in Coptis chinensis Franch.and C.teeta Wall.,respectively.In Coptis chinensis Franch.,the di-nucleotide repeat motifs of AG/CT were the predominant repeat types,a total of 953,accounting for 23.40% of the total SSR.The tri-nucleotide repeat motifs of AAG/CTT were the predominant repeat types,a total of 746,accounting for 18.3% of the total SSR;In C.teeta Wall.,the di-nucleotide repeat motifs of AG/CT were the predominant repeat types,a total of 934,accounting for 23.10% of the total SSR.The tri-nucleotide repeat motifs of AAG/CTT were the predominant repeat types,a total of 773,accounting for 19.10% of the total SSR.We randomly selected 60 pairs of primers for PCR amplification.Among them,12 pair primers showed polymorphism(20.00%).And 40 Coptidis plants were divided into 2 groups by using Populations software.There were numerous SSRs in Coptidis transcriptomes with high frequency and various types.It would provide abundant candidate molecular markers for genetic diversity analysis and genetic map construction of this plant. |
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