Transcriptome Sequencing And Differentlly Expressed Triterpenoid Synthesis Gene Analysis And Cloning Of Cyclocarya Paliurus Suspension Cells Induced By Aspergillus Niger Elicitor

Cyclocarya Paliurus(Bata1)Iljinskaja containing lots of bioactive secondary metabolites has been approved as a new food material,the resources shortage caused by the difficulties in breeding restricts its development and utilization.Triterpenoid is one of the important active component in it.In our laboratory,a stable cell suspension cultured cells to produce triterpene acid by using the plant cell culture technology has been established.Furthermore,the synthesis of triterpenoids by adding the exogenous elicitor additive into cultured cells were also promoted.And the mechanism of ANE(Aspergillus niger elicitor)from the signal transduction pathway and MAPK pathway were preliminarily explored,respectively.In order to explore the promotion mechanism of the synthesis of triterpenoids from ANE,the changes of antioxidant enzymes in the cultured cells of Cyclocarya paliurus under the inducing of ANE were studied.To investigate the biological mechanism of Cyclocarya paliurus in transcription level,the whole-transcriptome sequencing of cultured cells were performed using Illumina HiSeq4000 platform.Then,the differential expression genes of ANE-induced antioxidant system,regulators of secondary metabolic pathway,JA signal transduction pathway and triterpenoid metabolic pathway were analyzed comprehensively,and the key enzyme gene CpSS(Cyclocarya Paliurus squalene synthase)which were elaborated the molecular mechanism of triterpenoid regulation by ANE were cloned and conducted bioinformatics analysis.The main results are as follows:(1)The effect on the activity of antioxidant enzymes in the cultured cells of Cyclocarya paliurus induced by ANE: after 10 h induction of ANE,the triterpenoid content began to increase and reached the maximum value at 60 h,and the content increased from 12.47 ?g/mg to 36.46 ?g/mg,which was 3.36 times of the control group.Peroxidase(POD),superoxide dismutase(SOD),catalase(CAT),phenylalanine ammonia lyase(PAL)and phospholipase(PLC)were associated with the activity of antioxidant system,which showed obvious temporal changes.The activity of SOD showed three peak values at 1 h,8 h,66 h,respectively,and the activity of POD showed four peak values at 1h,8 h,21 h,66 h.With regards to the activities of CAT,there were five peak values at 2 h,8 h,24 h,5 4h and 66 h,and the activity of PAL had 3 peak values at 7 h,18 h and 42 h,respectively.The activity peak values of PLC appeared at 6 h and 24 h,which were all higher than the control group.(2)The transcriptome analysis of cultured cell introduced by ANE: In this study,the whole-transcriptome sequencing of three suspension cell samples(CK control group without ANE,ANE induced 20 h and 60 h)were carried out using Illumina HiSeq 4000 platform.In total of 67,230 high quality Unigenes with average length and N50 of 742 bp and 1379 bp were assembled.Then,Unigenes were performed sequence mapping usingBlastx tools with NR,Pfam,String,Swissprot,GO,COG and KEGG seven common databases.49.78% Unigenes were mapped into the public database,and 7,694 Unigenes were annotated to 25 COG functional categories.Furthermore,a total of 19,398 Unigenes obtained 128,396 GO annotation information.In addition,14,986 SSR sequences were obtained,which provides a abundant of information for further genetic analysis.(3)Differentially expressed genes of cells under the introduced-ANE: Through analyzing the differentially expressed genes,247,88,24,336 differentially expressed genes were found at 20 h and 60 h after induction compared to CK control group,respectively.Among these genes,20 POD,3 SOD and 1 CAT candidate genes associated with anti-oxidative system were up-regulated,9 JAZ,2 JAR and 12 MYC2 candidate genes related to JA signal transduction pathway were differentially expressed.15,990 candidate TF(transcription factor)genes were also identified,and there are a lot of TF gene family(bHLH,ERF,NAC,C2H2 and WRKY)involved in the regulation of triterpenoid metabolic pathway.A total of 75 candidate genes involved in terpenoid skeletal biosynthetic pathway and 22 genes involved in the triterpenoid biosynthetic pathway were identified.In addition,the RT-qPCR method was used to verify the expression of 12up-regulation genes related to triterpenoid metabolism.The results showed that the expression of these 12 genes was consistent with the result from whole-transcriptome sequencing.(4)CpSS full-length gene cloning and sequence analysis: In this paper,the candidate gene of squalene synthase was amplified according to whole-transcriptome sequencing of the cultured cells of Cyclocarya paliurus.Based on the full-length sequence,the sequence of CpSS gene was cloned as 1667 bp,which contained a complete ORF with 1245 bp encoding 414 amino acids using RT-PCR and RACE technologies.The highest similarity of CpSS was the walnut,and the similarity was up to 97%.The similarity of Grapes,soybeans,licorice,cocoa and other species were more than 85%.Sequence analysis showed that the protein of CpSS was unstable hydrophilic protein,which had the typical squalene synthase conserved region and domain and existed in the endoplasmic reticulum.It composed of multiple ?-helix cavitation structure,which was similar to the majority the plant squalene synthase protein structure.In conclusion,under the induction of ANE,the suspension cells of Cyclocarya paliurus have enhanced the expression of antioxidant enzymes in the oxidative stress system.Through mediating JA signal transduction pathways and regulating triterpenoid synthesis way of transcription factors,the key enzyme genes in the pathway are differentially expressed,thus promoting the synthesis and accumulation of triterpenoids in cells.