Biosynthesis Researches Of Secondary Metabolism In Arnebia Euchroma

Objective:Arnebia euchroma (Royle) Johnst is the main source for medicinal herb Lithospermum and Daodi herb of it. This study starting from A. euchroma environment, intends to investigate inducing effections of YE, Ag, Pb, Cd, Cr, low temperatures and UV-C to A. euchroma secondary metabolite production, especially to study biosynthesis amount rules of secondary metabolites with biological activity, such as phenolic compounds, furan compounds and shikonin compounds, etc. And from the genetic point of view, to build overexpression and RNA interference vectors of the key secondary metabolic pathway genes PAL, HMGR, PGT. This study cloned an AP2/ERF transcription factor family gene, which has a very significant role in the regulation on secondary metabolite and made annotations for it. At the same time, this thesis did systematically bioinformatics analysis on A. euchroma ERF subfamily based on local transcriptome. This paper put aims on building environmental and genetic research platform to guide biosynthesis of A. euchroma secondary metabolites.Method:①Using HPLC method to detect A. euchroma secondary metabolites content, of which are induced by YE, Ag, Pb, Cd, Cr, YE+Ag, YE+Pb, YE+Cd, YE+Cr,4℃low temperature and UV-C, and examine its time-based variation at the same time.②To clone A. euchroma PAL, HMGR, PGT gene, and transfer the total length of the target genes into over-expression vector pH7WG2D, and fragments of the coding region into RNA interference vector pK7GWIWG2D though the GATEWAY cloning technology.③Using the Lithospermum erythrorhizon AP2/ERF gene (ACX71873) as a probe sequence, a cDNA sequence of A. euchroma AP2/ERF named was cloned by in-silico-cloning method, and using local and online bioinformatics tools to do its annotations. ④Using the L. eiythrorhizon ERF gene(Genbank ID:261363612) as a probe sequence, to search local A. euchroma transcriptome database, combined with the NCBI database to separate A. euchroma ERF transcription factor candidate genes and do systematic annotation, molecular phylogenetic analysis and expression analysis.Result:①In the investigation effections of YE, Ag, Pb, Cd, Cr, YE+Ag, YE+Pb, YE+Cd, YE+Cr these nine elicitors on secondary metabolites of A. euchroma red cell suspension, it can be found that single heavy metals are more effective than YE and YE added elicitors, mainly as promoting role and especially24h at which is the most significant. The maximum promoting effect on secondary metabolites production of A. euchroma is achieved by Ag. The maximum content of the total phenolic acids, total LEFs, total shikonin and total secondary metabolites were all reached at48h in red suspension cells of A. euchroma and172%,199.2%,289%,205%higher than the control group respectively.Overall, the low temperatures show very significant promotion on total content secondary metabolites of A. euchroma red cell suspensions, but does not alter the overall change trend over time consistent with the control group. LEFs total content in12h,24h,48h,72h and168h group is73%,64%,84%,83%and42%higher than the control group respectively. However, the maximum appears at72h in induced group,24h in the control group.In the investigation effections of different times UV-C radiation on secondary metabolites in A. euchroma red callus suspension, at24h,48h,72h and168h, the content in induction group is26.4%,23.4%,21.4%and17%higher than the control group. The content of induction group is64.2%of control group at12h, indicating that after12h, the overall content of the induction groups start to exceed the control group. Overall, UV-C can promote the generation and accumulation of secondary metabolites of A. euchroma, and UV-C for5min at the168h has the most obvious effections. In the inspection on white lines of the A. euchroma suspension cells and callus cells, it can only detect phenolic compounds, the elicitor involved in experiment does not induce the synthesis of furan compounds or shikonin compounds. YE and heavy metals elicitor, cold and UV-C can raise total phenolic compounds content of white lines46.4%(168h, Cd),84.8%(4℃,24h) and92.6%(48h, UV-CV-15min) compared with the control group.②Download A. euchroma PAL, HMGR, PGT genes from NCBI, and using Primer primer5to design the over-expression, RNAi primers and corresponding primers containing CACC respectively. A. euchroma cDNA is used as template to clone full-length (the length followed by2086bp,2013bp,1023bp) genes which are overexpressing vector required and gene fragments (the length followed by490bp,525bp,328bp) which are RNAi vector required by PCR. Purified PCR products are connected to pGEM?-T Easy vector, hen use the high-fidelity enzyme Phusion? High-fidelity to do PCR and the products are transferred to the GATEWAY vector technology BP reaction entry vector pENTRTM/SD/D-TOPO? vector. After kanamycin resistance screening, the total length of the target genes are transfered into over-expression vector pH7WG2D, and fragments of the coding region into RNA interference vector pK7GWIWG2D. In addition, this study prepared Agrobacterium tumefaciens EH105competent cells for subsequent research.③A cDNA sequence of A. euchroma AP2/ERF named AeAP2/ERF was cloned by in-silico-cloning technology in this study, using ACX71873sequence from L. eiythrorhizon as the probe sequence. Bioinformatics results showed that the1077bp long gene includes a618bp ORF and encoding205amino acid. This gene encodes a protein of which instability factor is63.24, and might be an unstable acidic proteins. ProtScale results shows the end of the protein is significant hydrophilic. TMpred software predicted results show that the protein transmembrane helix region is not formed, indicating that the protein may not have transmembrane transporter signal. The results of AeAP2/ERF protein subcellular localization form Psort online software displaying that AeAP2/ERF is likely localized in the nucleus which has the maximum probability of70%. SignaIP4.1Server speculates that the protein encoded by AeAP2/ERF gene is a non-secreted protein and may not have signal peptide, it is synthesized in the cytoplasm but without protein transport. Through online software SOPMA and SWISS-MODEL to predict two and three protein structure of AP2/ERF respectively, the results show the maximum amount structure elements is the random coil, a-helices and extension stretch chain dispersed throughout the AeAP2/ERF protein. Phylogenetic analysis showed that AeAP2/ERF has the evolutionary shortest distance and closest relationship with L. erythrorhizon, and followed by Salvia in Lamiaceae. AeAP2/ERF gene encoded protein function analysis shows that the probability of the protein transcription regulation is8.8%, the probability of signal transduction is7.1%, and probability as the growth factor is4.6%. Since prediction probability of other features are too low, the AeAP2/ERF protein is likely to have a regulatory transcription and signal transduction functions.④This study isolated27ERF transcription factor family genes from A. euchroma transcriptome by BLASTp. Their size are between281bp and2758bp, the number of amino acids they encoded are distributing between78and390. Gene structure analysis of A. euchroma ERF show all AeERFs only exist a long exons, exons and non-coding regions vary in length, exon are between234and1173bp in length, the length of the non-coding region are between5and1834. All FPKM values of ERF genes expression level are between1and100. Physicochemical properties of the AeERFs protein are analyzed by Protparam, results show that this study involved A. euchroma ERF transcription factor genes are acidic. A. euchroma ERF transcription factor family genes overall are stable proteins. Since average of all AeERFs hydrophobic are negative, so all of then are the hydrophilic proteins. SOPMA and SWISS-MODEL results show that the maximum amount structure elements is the random coil, a-helices and extension stretch chain dispersed throughout the AeAP2/ERF protein. A. euchroma ERFs protein contains three conserved sequence:Motif1, Motif2and Motif3. Conserved sequence of Motif1is x[KR][KH][YF]RGVR[RQ]RPWGK[WFY][AV]AEIRDP, conserved sequence of Motif2is [RK] K[GR][AT]R[LV]WLGT[FY][ED]TAEEAA[LR]AYD[RK]AA[FLY][RK][LI][RK]GSKA[KL]L, conserved sequence of Motif3is NFPH[LE][VI]G.27proteins are mainly localized in the nucleus, micro, lysosomes, mitochondrial matrix or cell membranes. Furthermore building phylogenetic ML, NJ, ME method, UPGMA and MP tree for AeERFs, the results show AeERF transcription factor family is divided into two subfamilies,18proteins are belong to ERF and9are CBF/DREB subfamiliesConclusion:①In the case of genetic information does not change, to provide A. euchroma cell culture systems or transgenic general environmental conditions which are more similar to its Daodi estate area, namely low temperatures and high intensity ultraviolet radiation, can promote A. euchroma secondary metabolism, and improve the content of secondary metabolites. This study built environmental research platform and also the first study which concerned the impact of multi-factor and multi-level environmental factors on secondary metabolism of A. euchroma, this has important implications for the secondary metabolites biosynthesis study of A. euchroma.②From the perspective of genetic material, using GATEWAY technology to build key secondary metabolism gene overexpression vector and RNAi vectors A. euchroma, and bulid platform for constructions of related transgenic lines. Which is to build genetic material based genetic research platform, and that will provide a great convenience for digging and functional verificating the genes on secondary metabolic pathway, and also fill the gaps in in transgenic research of A. euchroma.③In-silico-cloning is a new cloning technique which based on sequences splicing and assembing, can provide an accurate reference sequence for experimental cloning. This study can lay a foundation for future experiments cloning of A. euchroma AP2/ERF family gene, function identification, genetic engineering and the application of this technique in traditional Chinese medicine. This study palys a role in building bioinformatics-based genetic research platform, and also provides a reference for the wide use of in-silico-cloning technology in the field of traditional Chinese medicine.④Transcriptome based bioinformatics studies can get more useful biological information and more efficient. In this study, local transcriptome data combing with common public database, we separate a transcription factors family which regulats the secondary metabolism, and make a comprehensive bioinformatics analysis for the family. This study palys a role in building bioinformatics-based genetic research platform, and of great significance for exploring the genetic mechanisms of biological secondary metabolites, and also fill gaps in bioinformatics research of non-model plant.