Study On Biosynthesis And Regulation Of Anthraquinone In Cassia Obtusifolia By Hybrid Sequencing

Cassia obtusifolia is a leguminosae plant,and its seed was called semen cassiae torae.It was a commonly used traditional Chinese medicine and was included in the Chinese Pharmacopoeia.C.obtusifolia contains a lot of anthraquinone compound such as aurantio obtusin and emodin.However,there are few reports on the biosynthesis and regulation of anthraquinone compound in C.obtusifolia.In this paper,HPLC was used to determine the content of aurantio obtusin and emodin in different tissues of C.obtusifolia.It was found that aurantio obtusin and emodin are mainly enriched in C.obtusifolia seed.This provides a basis for the study of biosynthesis and regulation mechanisms of anthraquinones in different tissues by high-throughput sequencing.Next,we carried out the joint sequencing of the second and third generation transcriptomes of Cassia obtusifolia.The root,stem,leaf,flower and seed of C.obtusifolia were sequenced in the second generation,and the five tissues were mixed into a sample F01 for sequencing in the third generation.The second-generation transcriptome sequencing of each sample produces no less than 6Gb of Clean Data,ensuring that Q30 reaches 85%.After comparison with 8 functional databases,98.25%(57092)genes were found to have homology.In the GO classification annotation,the number of genes involved in the metabolic process is 21,114.A total of 1,214 transcripts involved in the biosynthesis of secondary metabolites were found in the KEGG functional classification.The number of transcripts involved in the biosynthesis of phenylpropanoids,steroids,and terpenoids were: 363,79,165.These transcripts provide precursors for the biosynthesis of anthraquinones.A total of 4,315,774 subreads were obtained from F01 sample,601,168 polymerase sequences,282,022 ROI sequences,136,567 full-length non-chimeric sequences,114185 non-full-length sequences,67722 consensus sequences.F01 obtained 58,106 de-duplication sequences,and 40,118,41,711,4,388,82,771,45,922 transcripts were found in the seed,root,stem,leaf,and flower of C.obtusifolia.With FPKM >10 as the threshold,the number of genes with higher expression abundance in root,stem,leaf,flower,seed was 7,877,9,023,8,114,9,082 and 5,870.The number of genes specifically expressed in root,stem,leaf,flower and seed of C.obtusifolia was 714,432,889,2,629 and 658.In addition,based on the de-redundant sequence of the sample,F01 predicted 2,712 variable shear events,and the analysis predicted 40,160 SSR,37,659 intact CDS,and 775 lncRNA.Analysis of C.obtusifolia Transcriptome data revealed several genes that may be involved in the biosynthesis of anthraquinones,involving the MVA pathway,MEP pathway,shikimate pathway,and polyketide pathway.It is speculated by differential expression analysis and qRT-PCR that ICS,DXS and IPPS may be important regulatory targets in the biosynthesis pathway of anthraquinones.The CYPs gene,which may be involved in the biosynthesis of anthraquinones,together with 85 CYPs genes from other plants to construct a phylogenetic tree,suggesting its possible biological functions.303 transcription factors related to the formation and development of seed were screened,including 101 ZF,24 MYB,24 ARF,19 NAC,18 Homeobox,18 bHLH and 15 NF,passed RT-qPCR and GO analysis were validated and further functional clustering.The paper also analyzed the expression pattern of eight Hsp20 genes may be related to stress resistance of C.obtusifolia.This provides the transcriptome data and theoretical basis for the systematic study of the genes related to the biosynthetic pathway of anthraquinones,the genes related to the seed formation and development of C.obtusifolia and the genes related to the stress resistance of C.obtusifolia.This paper also study the function and expression pattern of CoDXS and CoDXR genes in the biosynthetic pathway of anthraquinones.The paper completed the cloning of OFR according to the sequence of functional annotation of CoDXS and CoDXR in C.obtusifolia transcriptome,the prokaryotic expression vectors pTrc-CoDXS and pTrc-CoDXR were separately expressed in TOP10,and the function of CoDXS and CoDXR genes were verified by plaque color change of E.coli TOP10 and HPLC detection content of ?-carotene.At the same time,the expression pattern of CoDXS and CoDXR genes in different tissue and stress condition were resarched.The results showed that the expression of CoDXS and CoDXR genes showed different trend in different tissue and different stress condition.This may be due to differences in the mechanisms of receiving,transmitting and responding to signals in plants involved in CoDXS and CoDXR genes under different tissues and stress conditions.This result lay a foundation for further study on the role of CoDXR and CoDXS genes in regulating stress and the regulation of related genes in the MEP biosynthesis pathway of anthraquinones.