基于多组学分析的丹参酚酸类成分生物合成与抗盐性调控研究

【作者】 韩立敏；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2020， 博士

【摘要】 丹参(Salvia miltiorrhiza Bunge)为唇形科鼠尾草属常用药用植物,以其干燥的根或根茎入药,水溶性酚酸类次生代谢产物为丹参重要的药用活性成分之一,具有抗氧化、抗菌、消炎、抗肿瘤等多种药理活性。虽然丹参酚酸类成分的生物合成途径备受关注,但其调控机制仍不是特别清楚。据报道,野生丹参比栽培丹参酚酸含量高、抗逆性强,本研究基于丹参基因组、野生与栽培丹参比较代谢组及转录组联合分析,重点筛选丹参酚酸类合成相关转录调控因子和抗逆调节基因,并采用遗传操作技术研究基因的功能,为深入了解酚酸类有效成分生源合成途径及调控机制、揭示环境因素对次生代谢产物形成与积累的影响提供了理论依据。主要研究结论如下:1、基于UPLC-MS/MS检测技术的丹参广靶代谢组学研究对野生丹参与栽培丹参进行了代谢组学测定与分析,共检测到658种代谢物,其中差异代谢物286种。比较代谢组学分析结果表明,在根比较组中,差异显著代谢物共159种,酚酸类成分中丹酚酸C的差异最为显著。差异代谢物KEGG富集分析显示,酚酸类化合物合成途径、黄酮类化合物合成途径以及两者的上游通路苯丙素生物合成途径上的差异代谢物是造成野生与栽培丹参差异的化学基础。相关性分析结果表明,丹酚酸B与丹酚酸C的含量呈显著正相关,提示两者可能共享相同的代谢调控机制,丹酚酸C可能作为丹酚酸B合成的前体物质参与酚酸类成分的生物合成。2、基于Illumina测序的丹参转录组学分析对野生与栽培丹参进行了转录组测序,共获得88.64 Gb Clean Data,注释30,286条Unigene。比较转录组学分析结果显示,差异表达基因主要与植物代谢、响应刺激、植物抗逆、铵态氮运输和抗氧化有关,其是导致野生与栽培丹参抵抗逆境、次生代谢物合成以及营养吸收能力差异的遗传基础。酚酸类成分合成途径关键酶基因在栽培丹参根中的表达量普遍高于野生丹参根,在叶片中的表达量则低于野生丹参叶中的表达量;黄酮类合成途径酶基因的表达表现出相同的变化趋势。抗氧化、抗逆相关差异基因中,SOD的表达在野生与栽培丹参中呈显著差异。3、丹参转录组学与代谢组学的关联分析通过代谢物与基因表达的关联分析,筛选到了丹酚酸C合成相关的细胞色素P450家族基因21个、漆酶编码基因11条。筛选到丹酚酸C合成调控相关的转录因子有 16个MYB、23个bHLH、13个WRKY、23个ERF、10个NAC、4个TIFY、4个热胁迫转录因子、4个Trihelix、2个bZIP等。关联到的旁支木质素代谢途径与黄酮类合成途径关键酶基因均与丹酚酸C的含量显著负相关。4、SmMYB13与SmMYB70对丹参酚酸类和黄酮类次生代谢的调控功能研究克隆了丹酚酸C合成相关转录因子SmMYB13和根中特异表达转录因子SmMYB70。SmMYB13和SmMYB70均响应机械损伤、脱水、硫酸铜、低温、NaCl、PEG8000、赤霉素,以及茉莉酸甲酯处理。过表达SmMYB13与SmMYB70均会提高丹参总酚酸和总黄酮的含量。在SmMYB13过表达株系中,酚酸类合成途径SmPAL2、Sm4CL1、SmC4H、SmHPPR、SmRAS、SmCYP98A14 表达量显著上调,黄酮类合成途径酶基因SmCHS1、SmCHS2、SmFLS1、SmFLS2的表达量显著提高;在SmMYB70过表达株系中,酚酸类合成途径SmPAL2、Sm4CL1、SmC4H、SmHPPR、SmCYP98A14显著上调,SmPAL1、Sm4CL2、SmTAT显著下调,SmRAS变化不显著,黄酮类合成途径上游基因整体被上调、下游基因整体被下调。上述结果提示SmMYB13与SmMYB70在丹参酚酸类和黄酮类次生代谢调控中发挥重要作用。5、SmMYB13与SmMYB70过表达丹参抗逆的分子机制初探研究表明,MYB转录因子可通过调控SOD基因的表达影响植物对多种生物与非生物胁迫的响应。本研究中,基于丹参基因组系统鉴定并分析了 SmSOD基因家族。分别过表达SmMYB13与SmMYB70均提高了该基因家族大部分成员的表达量,SOD总酶活性升高,盐胁迫下细胞中的丙二醛含量低于非转基因株系,两个基因的过表达都可以提高丹参对盐胁迫的耐受性。综上,本研究基于多组学联合分析,分析了野生丹参与栽培丹参的差异代谢物和差异表达基因,并重点研究了筛选的两个MYB转录因子的功能,初步阐明了丹参应对盐胁迫逆境的分子机制。该研究对深入解析酚酸类成分的生物合成途径和调控机制,培育有效成分含量高、抗逆性强的优质丹参品种具有重要意义。更多还原

【Abstract】 Salvia miltiorrhiza Bunge is a common medicinal plant of Salvia in Labiatae.Its dried roots or rhizomes are used as traditional chinese medicine.Water-soluble phenolic acids are one of major groups of active ingredients which responsible for the pharmacological effects of S.miltiorrhiza.Phenolic acids have many pharmacological activities such as antioxidant,antibacterial,anti-inflammatory,anti-tumor,and so on.Although the biosynthetic pathway of salvianolic acids has attracted much attention,it has not been fully elucidated,and the regulatory mechanism is still unclear.It is reported that wild S.miltiorrhiza has higher phenolic acids and stronger stress resistance than cultivated S.miltiorrhiza.Here,based on the integration of genome,comparative metabolomics and transcriptomics of wild and cultivated S.miltiorrhiza,we focused on the screen of transcription factors related to phenolic acids biosynthesis and genes related stress resistance,and used genetic manipulation technology to study the function.Our study laid a solid theoretical foundation for deeply understanding the biosynthesis and regulatory mechanism of phenolic acids,and revealed the influence of environmental factors on the formation of secondary metabolites.The main conclusions are as follows.1.Broad targeted metabolomics of S.miltiorrhiza based on UPLC-MS/MS.The metabolomics of wild and cultivated S.miltiorrhiza were determined and analyzed.A total of 658 metabolites were detected and 286 metabolites with significant difference were obtained.The results of comparative metabolomics analysis showed that there were 159 metabolites with significant difference in root comparative group.Among the differential phenolic acids,the change of salvianolic acid C was the most significant.KEGG enrichment analysis implied that the differential metabolites in the biosynthetic pathway of phenolic acids,flavonoids and phenylpropanoids were the chemical basis of the differences between wild and cultivated S.miltiorrhiza.The metabolites correlation analysis results demonstrated that the content of salvianolic acid B and C exhibited significantly positive correlated with each other,suggesting that they may share the same metabolic regulation mechanism,and salvianolic acid C may participate in the biosynthesis of phenolic acids as a precursor of salvianolic acid B.2.Transcriptome analysis of S.miltiorrhiza based on Illumina sequencingTranscriptome sequencing of wild and cultivated S.miltiorrhiza was carried out.A total of 88.64 GB clean data were obtained and 30,286 Unigenes were annotated.Comparative transcriptome analysis indicated that the function of differentially expressed genes in wild and cultivated S.miltiorrhiza was mainly related to plant metabolism,response to stimulus,plant stress resistance,and antioxidant activity,which may be the genetic basis for the differences in stress resistance,secondary metabolite biosynthesis and nutrient absorption capacity of wild and cultivated S.miltiorrhiza.The expression levels of key enzyme genes in phenolic acid biosynthetic pathway of cultivated roots were generally higher than those in wild roots,while the expression levels in leaves were lower than those in wild leaves.Expression of flavonoid biosynthetic pathway enzyme genes showed the same trend.Among the genes related to antioxidant and stress resistance,the expression of SODs showed significant difference in wild and cultivated S.miltiorrhiza,indicating the critical role in the response of stress.3.Integration analysis of transcriptomics and metabolomics of S.miltiorrhizaThrough the correlation analysis of metabolites and gene expression,21 cytochrome P450 family genes and 11 laccase coding genes related to salvianolic acid C biosynthesis were screened,including 16 MYBs,23 bHLHs,13 WRKYs,23 ERFs,10 NACs,4 TIFYs,4 heat stress transcription factors,4 trihelix,and 2 bZIP.The key enzyme genes in lignin metabolism and flavonoid biosynthesis were negatively correlated with the content of salvianolic acid C.4.Function of SmMYB13 and SmMYB70 in the regulation of salvianolic acid and flavonoid biosynthesisSmMYB13,a transcription factor related to salvianolic acid C biosynthesis,and SmMYB70,a transcription factor specifically expressed in roots,were cloned.Both SmMYB13 and SmMYB70 response to wound,dehydration,CuSO4,chilling,NaCl,PEG8000,GA,and MeJA treatment.Overexpression of SmMYB13 and SmMYB70 increased the contents of total phenolic acids and total flavonoids in S.miltiorrhiza.In the SmMYB13 overexpressed transgenic plants,the expression of SmPAL2,Sm4CL1,SmC4H,SmHPPR,SmRAS,and SmCYP98A14 in phenolic acids biosynthetic pathway were significantly up-regulated,and the expression of SmCHS1,SmCHS2,SmFLS1,and SmFLS2 in flavonoids biosynthetic pathway were significantly increased.In the SmMYB70 overexpressed transgenic plants,the expression of SmPAL2,Sm4CL1,SmC4H,SmHPPR,and SmCYP98A14 in phenolic acids biosynthetic pathway were significantly up-regulated,and SmPAL1,Sm4CL2,and SmTAT were significantly down-regulated,while SmRAS was not significantly changed.The upstream genes in flavonoids biosynthetic pathway were up-regulated and downstream genes were down regulated.These results suggested that SmMYB13 and SmMYB70 play an important role in the regulation of phenolic acids and flavonoids.5.Molecular mechanism of SmMYB13 and SmMYB70 overexpressed S.miltiorrhiza under stressStudies have shown that MYB transcription factor can affect plant response to various biotic and abiotic stresses by regulating the expression of SOD.The SmSOD gene family related to stress tolerance was systematically identified and analyzed in the genome wide.Overexpression of SmMYB13 and SmMYB70 respectively promoted the expression of most SmSODs,and the total SOD activity was elevated.Under salt stress,the MDA content in cells of transgenic lines was lower than that in non-transgenic lines.The overexpression of SmMYB13 and SmMYB70 enhanced the tolerance of S.miltiorrhiza to salt stress.In conclusion,the differential metabolites and differentially expressed genes between wild and cultivated S.miltiorrhiza were analyzed based on the integrated analysis of multiple omics.The function of two screened MYB transcription factors were studied,and the molecular mechanism of S.miltiorrhiza in response to salt stress was preliminarily clarified.This research is of great significance for comprehensively elucidating the phenolic acids biosynthetic pathway and its regulatory mechanism,and for breeding the S.miltiorrhiza with high content and strong stress resistance.更多还原