丹参迷迭香酸合成途径相关基因的功能研究

【作者】 宋婕；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2010， 博士

【摘要】 丹参(Salvia miltiorrhiza Bunge)为唇形科多年生草本植物,根入药,具有抗氧化、抗病毒、抗肿瘤等活性,在临床上可用于心脑血管疾病、癌症及各种炎症的治疗。近年来,伴随着丹参药材需求的日益扩大和野生资源的逐渐减少,提高丹参药材活性成分的含量、培育优质新品种已成为丹参资源开发中亟待解决的关键问题之一。丹参的活性物质分为两大类：一类为水溶性的酚酸类物质,包括咖啡酸、丹参素、迷迭香酸、丹酚酸、紫草酸等；另一类为脂溶性的丹参酮类化合物,包括丹参酮Ⅰ、丹参酮ⅡA、隐丹参酮等。鉴于传统中药以水煎服的用药方式,丹参的水溶性成分逐渐成为近年来研究的热点。迷迭香酸是丹酚酸B等复杂酚酸类活性物质的核心结构单元,丹酚酸B是由迷迭香酸衍生而来。苯丙烷类代谢途径和酪氨酸代谢途径共同参与了迷迭香酸的生物合成。目前该途径上的苯丙氨酸解氨酶(PAL)、肉桂酸-4-羟化酶(C4H)、4-香豆素辅酶A连接酶(4CL)、酪氨酸氨基转移酶(TAT)、羟基苯丙酮酸还原酶(HPPR)等多数酶基因已被克隆,但这些酶基因的调控基因还不清楚,各个酶基因对迷迭香酸积累贡献的大小也没有系统的报道。本论文在已有研究的基础上,进一步克隆参与丹参迷迭香酸生物合成的相关酶和转录因子基因,并以其中的酶基因为主要研究对象,分析这些基因的表达与迷迭香酸及其衍生的丹酚酸B等酚酸类成分积累之间的相关性,利用RNAi的方法探讨这些酶基因在迷迭香酸和丹酚酸B合成过程中的作用,筛选影响丹参迷迭香酸合成的关键基因,为丹参次生代谢物质的调控、分子育种及品质成因等研究奠定基础。主要研究内容及结论如下：1.利用PCR方法在丹参中克隆获得一条R2R3-MYB类转录因子基因MYB4, Genbank注册号为GU586494。该基因由两个外显子和一个内含子组成,包含一个长为693bp的开放阅读框(ORF),编码230个氨基酸。氨基酸序列含有两个保守的MYB DNA结合结构域以及MYB4类转录因子的保守基序LNLDL,且与拟南芥中C4H因的负调控因子MYB4和MYB32具有较高的相似性。实时荧光定量PCR分析结果显示,丹参MYB4基因在根、茎、叶中均有表达,但在叶中表达量最高。茉莉酸甲酯(MeJA)可以在一定程度上抑制该基因的表达,而光照和脱落酸(ABA)则可以诱导其表达。不同部位及不同处理阶段丹参MYB4基因表达的变化趋势与C4H基因相反,推测丹参MYB4转录因子可能作为C4H基因的转录抑制子发挥作用。2.克隆了一条酰基转移酶基因家族成员迷迭香酸合酶类似基因(RAS-like)及其5’侧翼序列,Genbank注册号分别为GU64719、GU647200。该基因开放阅读框长为1284bp,编码一个由427个氨基酸组成的蛋白,含有BAHD酰基转移酶家族的保守基序HXXXD和DFGWG,与丹参同科植物紫苏中报道的迷迭香酸合酶(RAS)具有较高的相似性。RAS-like基因在丹参的根、茎、叶中均有表达,其中茎中表达量最高。该基因的启动子序列含病原菌诱导响应元件Box W1、MeJA响应元件以及多种光作用元件。进一步利用实时荧光定量PCR分析发现RAS-like基因的表达可以受黄瓜细菌性角斑病菌、MeJA,光照、水杨酸(SA)的诱导。通过RNAi方法降低丹参中RAS-like基因的表达可以引起迷迭香酸及其衍生的丹酚酸B含量的降低,但干涉株系中总酚和总黄酮含量以及抗氧化活性变化不显著,推测RAS-like基因可能编码·迷迭香酸合酶参与丹参迷迭香酸的生物合成。3.利用DNA Walking方法克隆了丹参C4H基因的5’侧翼序列,Genbank注册号为GQ896332。在此基础上进一步分析了丹参迷迭香酸途径相关酶基因(PAL、C4H、4CL、TAT, HPPR, RAS-like基因)启动子区域的顺式作用元件,并对不同基因启动子序列的顺式作用元件进行了归类比较。结果显示,光响应元件是存在最多且最普遍的作用元件。其次,MeJA响应元件和ABA响应元件以及MYB结合位点也在多数基因的启动子区域存在,由此推测光照、MeJA、ABA、MYB转录因子等因素可以通过同时调节迷迭香酸途径相关酶基因的表达来影响迷迭香酸及其衍生的丹酚酸B的生物合成。4.利用实时荧光定量PCR和高效液相色谱技术分别检测了光照处理和MeJA处理后迷迭香酸合成相关酶基因的表达变化以及对应条件下迷迭香酸和丹酚酸B的含量。在此基础上,采用典型相关分析的统计方法,将丹参迷迭香酸代谢途径相关酶基因的表达和目标代谢物的积累水平进行整合分析,构建了“基因表达-代谢物积累”关联谱。对基因与产物的相关性分析发现,PAL1、PAL2、C4H、4CL2、HPPR基因是光照条件下迷迭香酸合成的关键基因。PAL1、 C4H、HPPR基因是光照条件下丹酚酸B合成的关键基因。5.利用RNAi方法降低PAL、C4H、4CL、TAT、HPPR等迷迭香酸途径相关酶基因的表达,分析各基因在丹参酚酸类物质积累及抗氧化过程中的作用。结果显示,抑制上述基因的表达均能在不同程度上降低丹参总酚和总黄酮的含量以及抗氧化活性,说明上述各基因在丹参的酚酸类成分代谢中均发挥着重要作用。此外,抑制上述各基因的表达均能降低丹参中迷迭香酸和丹酚酸B的含量,其中,T4T基因的影响最为显著,其次是PAL基因。对各基因干涉株系进一步比较分析发现,在非诱导条件下,PAL1、4CL2和RAS-like基因是迷迭香酸生物合成的关键基因,而TAT基因则在迷迭香酸和丹酚酸B的合成过程中均发挥关键作用。更多还原

【Abstract】 Medicinal Salvia miltiorrhiza ("Danshen" in Chinese) is a perennial plant which belongs to Labiatae family. As a traditional medicine, its roots have important biological activities, including antioxidant, antitumor, and antimicrobial properties."Danshen" is renowned for its curative effects on coronary heart diseases, particularly angina pectoris and myocardial infarction. In recent years, accompanied by the growing demand for Danshen and a gradual reduction of its wild resources, to improve the content of the active ingredients and cultivate new varieties with high quality have become the most urgent and key problems in the development of Salvia resources. The active pharmaceutical ingredients of S. miltiorrhiza are divided into two main groups:water-soluble phenolic acids, such as caffeic acid, danshensu (3,4-dihydroxyphenyllactic acid), rosmarinic acid, salvianolic acids and lithospermic acid, and lipid-soluble tanshinones such as tanshinone I, tanshinone IIA and cryptotanshinone. The phenolics now attract more attention because they are the main components of water decoction, which is the most common form of dosing administered to patients in Chinese clinics. Rosmarinic acid is thought to be the core structure of most hydrophilic compounds in S. miltiorrhiza, such as salvianolic and lithospermic acids. A proposed biosynthetic pathway in Coleus blumei suggests that rosmarinic acid is an ester of3,4-dihydroxyphenyllactic acid and caffeic acid. Those two compounds are synthesized via the tyrosine-derived pathway and the phenylpropanoid pathway, respectively. At present, genes encoding the enzymes in the rosmarinic acid biosynthesis pathway including phenylalanine ammonia-lyase (PAL), cinnamate4-hydroxylase (C4H),4-coumarate:coenzyme A ligase (4CL), tyrosine amino-transferase (TAT) and hydroxyphenylpyruvate reductase (HPPR) have been cloned in S. miltiorrhiza. But the regulation genes of these enzymes are still unclear. The reports for the contribution of these genes to rosmarinic acid accumulation are also lacking. In this study, to gain insight into the nature of rosmarinic acid biosynthesis, we further cloned the genes which encoded the enzymes and transcription factors in the rosmarinic acid pathway in S. miltiorrhiza. The relationship between the expression levels of these genes and the accumulations of rosmarinic acid and salvianolic acid B were also analyzed. Furthermore, RNAi was used to study the function of the enzyme genes in the biosynthesis of the active water-soluble phenolic acids. Moreover, the key genes for the synthesis of rosmarinic acid and its derivatives were screened, which is of great significance for the further studies of the regulation of the secondary metabolites and molecular breeding of S. miltiorrhiza. The main results and conclusions are as follows:1. The entire sequence named MYB4, which belonged to R2R3-MYB transcription factor gene family, was cloned in S. miltiorrhiza by PCR. The Genbank accession number was GU586494. MYB4in S. miltiorrhiza consisted of two exons and one intron, and contained an open reading frame (ORF) of693bp length that encoded a protein of230amino acids. The amino acid sequence contained two conserved MYB DNA-binding domains, as well as the conserved motif of MYB4transcription factor (LNLDL). Sequence analysis showed that it shared high identity with MYB4and MYB32in Arabidopsis thaliana, which were two negative regulatory factors for C4H. The expression pattern of MYB4gene was analyzed by real-time quantitative PCR. The results indicated that it expressed in all S. miltiorrhiza organs but most highly in leaves. Methyl jasmonate (MeJA) could inhibit the expression of MYB4, while light and abscisic acid (ABA) can induce its expression in S. miltiorrhiza. MYB4shared contrary expression pattern with C4H at different stages of these treatments, suggesting that the transcription factor encoded by MYB4may function as the repressor of C4H in S. miltiorrhiza.2. RAS-like gene and its5’flanking sequence were cloned in S. miltiorrhiza by degenerated PCR and DNA Walking method. The Genbank accession numbers were GU647199and GU647200respectively. RAS-like gene contained an ORF of1284bp length encoding a protein of427amino acids with typical characteristics (conserved HXXXD motif and DFGWG motif) of the BAHD acyltransferase superfamily. It shared high identity with the rosmarinic acid synthase (RAS) in C. blumei. RAS-like gene expressed in roots, stems and leaves of S. miltiorrhiza but most highly in stems. Totally,900bp5’flanking region of RAS-like gene was obtained and the putative cis-elements in this region including pathogen responsive element (Box Wl), element involved in the MeJA-responsiveness, and a variety of light responsive elements were predicated. Based on this, the expression pattern of RAS-like gene was analyzed by real-time quantitative PCR. The results showed that the expression of RAS-like gene could be induced by Psoudomonas lachrymans, MeJA, light and salicylic acid (SA). Suppressing of RAS-like gene in S. miltiorrhiza via RNAi can lead to the reduction of rosmarinic acid and salvianolic acid B. But the DPPH radical scavenging activity and the contents of total phenolics and total flavonoids did not change significantly. These results indicated that RAS-like gene may play a role in the biosynthesis of rosmarinic acid in S. miltiorrhiza as the rosmarinic acid synthase gene.3. The5’flanking sequence of C4H was cloned in S. miltiorrhiza by DNA Walking. The Genbank accession number was GQ896332. Based on this, the cis-acting elements in the promoter regions of the rosmarinic acid-related genes(PAL, C4H,4CL, TAT, HPPR and RAS-like gene) in S. miltiorrhiza were analyzed, compared and classified. The results showed that light responsive elements were the most abundant elements and existed in the promoter regions of all rosmarinic acid-related genes. MYB-binding site and elements involved in MeJA and ABA responsiveness also existed in the promoter regions of the majority genes. These results indicated that light, MeJA, ABA and MYB transcription factors can influence the accumulation of rosmarinic acid and its derivatives by regulating the enzyme genes in the rosmarinic acid pathway at the same time.4. Gene expression levels and the accumulation of rosmarinic acid under the treatment of light or MeJA were detected by real-time quantitative PCR and high performance liquid chromatography respectively. Furthermore, a "gene-to-metabolite" network was constructed according canonical correlation analysis. By analyzing the relation between gene expression and metabolite accumulation, we found that PALI, PAL2, C4H,4CL2and HPPR were the key genes for rosmarinic acid biosynthesis under light treatment; PALI, C4H and HPPR were the key genes for the biosynthesis of salvianolic acid B under light treatment.5. Expression of PAL, C4H,4CL, TAT and HPPR were silenced in S. miltiorrhiza by RNAi to discuss the function of these genes in the process of antioxidant and the accumulation of water-soluble phenolic acids. The results showed that suppressing the expression of each gene can reduce the DPPH radical scavenging activity and the contents of total phenolics and total flavonoids, indicating that all the above genes played important roles in the metabolic course of the active ingredients in S. miltiorrhiza. The contents of rosmarinic acid and salvianolic acid B in different RNAi lines were also detected. Among them, the influence of TAT was the most significant, followed by PAL. After comparing between the RNAi lines for different genes, we concluded that PALI,4CL2and the RAS-like gene cloned here were the key genes for rosmarinic acid biosynthesis under non-induced condition. While TAT was the key gene for the biosynthesis of both rosmarinic acid and salvianolic acid B under non-induced condition.更多还原