药学学报  2020, Vol. 55 Issue (9): 2110-2121     DOI: 10.16438/j.0513-4870.2020-0429   PDF    
丹参酮和丹酚酸类化合物的生物合成及其转录调控机制
詹忠根, 李杏     
浙江经贸职业技术学院生物制药教研室, 浙江 杭州 310018
摘要: 丹参酮和丹酚酸类化合物是治疗冠心病、心肌梗死、高血压、高血脂、脑卒中等疾病的重要物质基础。近年来,随着基因组学、转录组学、代谢组学和生物信息学的快速发展,其生物合成途径和转录调控机制研究取得较大进展。文章在总结丹参酮和丹酚酸类化合物生物合成最新研究的基础上,着重综述了转录因子对其生物合成的调控作用,认为进一步厘清丹参酮和丹酚酸类化合物的生物合成途径(尤其是下游合成途径的研究)和不同家族转录因子的相互作用、信号响应和协同调控机制,对于挖掘其合成、转运、调节、修饰相关的新基因及揭示调控网络的分子机制十分必要;同时,根据丹参活性成分生物合成规律,人工设计并构建新的、具有特定生理功能的生物系统,从而大幅度提高合成酶基因的表达及活性物质的产量,是有待进一步深入研究的方向之一。
关键词: 丹参酮类化合物    丹酚酸类化合物    生物合成途径    转录因子    
Biosynthesis and transcriptional regulation of tanshinones and salvianolic acids
ZHAN Zhong-gen, LI Xing     
Biophamaceutical Laboratory, Zhejiang Institute of Economics and Trade, Hangzhou 310018, China
Abstract: Tanshinones and salvianolic acids are important materials in the treatment of coronary heart disease, myocardial infarction, hypertension, hyperlipidemia, stroke and others illnesses. In recent years, with the development of genomics, transcriptome, metabolomics and bioinformatics, many advances have been made in the biosynthesis and transcriptional regulation of tanshinones and salvianolic acids. This paper summarizes these advances and suggests further study on the downstream synthesis pathways and transcriptional regulatory mechanisms to reveal new molecular mechanism of synthesis, transport, regulation and modification. Additionally, we discuss the design and construction of new biological pathways to increase the expression of biosynthesis genes and the production of secondary components, is a newly developing research field.
Key words: tanshinone    salvianolic acid    biosynthesis pathway    transcription factor    

中药丹参为唇形科(Labiatae)鼠尾草属(Salvia)植物丹参(Salvia miltiorrhiza Bge.)的干燥根及茎, 含有丰富的丹参酮类化合物, 如丹参酮Ⅰ (tanshinone Ⅰ)、丹参酮ⅡA (tanshinone ⅡA)、丹参酮ⅡB (tanshinone ⅡB)、二氢丹参酮Ⅰ (dihydrotanshinone Ⅰ)、隐丹参酮(cryptotanshinone)等(图 1)和丹酚酸类化合物, 如丹参素(3, 4-dihydroxyphenyllactic acid)、咖啡酸(caffeic acid)、迷迭香酸(rosmarinic acid)、丹酚酸A (salvianolic acid A)和丹酚酸B (salvianolic acid B)等(图 2), 具有活血化瘀、消肿止痛、养心安神之功效, 是治疗冠心病、心肌梗死、高血压、高血脂等疾病的主要物质基础[1]。然而天然资源中丹参酮和丹酚酸类化合物含量极低, 且其累积与地理位置、生态环境及遗传背景有关, 也受诱导子、关键酶、调节基因或转录因子的调控。为充分利用生物技术方法, 有效提高丹参酮和丹酚酸等药效成分含量, 近年来, 测序和解析了丹参全基因组、叶绿体和线粒体[2, 3], 发表了20多个来自丹参不同器官及生长阶段的转录组数据[4, 5], 以及克隆与鉴定了大部分关键酶基因[6, 7], 为阐明丹参酮和丹酚酸类化合物的生物合成途径, 揭示转录因子单独或协同调控机制作出了极大贡献。前人已经对丹参酮和丹酚酸类化合物的生物合成、诱导子的诱导与调控等进行了详细的总结[6-8], 本文主要介绍该类化合物生物合成的最新研究, 着重综述转录因子的调控作用。

Figure 1 Representative compounds of tanshinones

Figure 2 Representative compounds of phenolic acids
1 丹参主要药效成分的生物合成研究 1.1 丹参酮类化合物的生物合成

丹参酮类化合物属于二萜类, 主要在丹参根部周皮合成和积累, 迄今已分离到50余种。其生物合成包括牻牛儿基牻牛儿基焦磷酸(GGPP)合成、GGPP环化和次丹参酮二烯修饰等3个阶段(图 3)。GGPP的合成与其他天然二萜类基本一致, 即甲羟戊酸途径(MVA)合成的异戊烯基焦磷酸(IPP)与甲基赤鲜醇4-磷酸途径(MEP)合成的二甲基烯丙基焦磷酸(DMAPP)在法呢基焦磷酸合酶(SmFPPS)、牻牛儿基牻牛儿基焦磷酸环化酶(SmGGPPS)作用下生成GGPP。但不同于其他天然二萜类的是丹参酮生物合成中, GGPP的合成前体主要来源于MEP途径[9]。GGPP环化阶段, 先由柯巴基焦磷酸合酶(SmCPS)催化GGPP转化成柯巴基焦磷酸(CPP), 再由类贝壳衫烯合酶(SmKSL)将CPP环化成次丹参酮二烯(miltiradiene)[10, 11]。在此过程中, SmCPS的作用非常重要, 直接决定次丹参酮二烯碳架结构的形成。抑制SmCPS1的表达, 丹参酮ⅡA和丹参酮Ⅰ的合成显著下降[10]。而SmKSL之所以能催化CPP生成次丹参酮二烯, 这一有别于裸子植物中紫杉醇、银杏内酯等生物合成过程的新功能, 得益于其编码基因SmKSL在进化过程中缺失了‘internal/γ’结构域[11]。次丹参酮二烯的修饰, 主要包括细胞色素P450家族蛋白SmCYP76AH1催化次丹参酮二烯生成铁锈醇(ferruginol)[12], SmCYP76AH3羟化铁锈醇的C-7和C-11位生成11-羟基铁锈醇(11-hydroxy ferruginol)、柳杉酚(sugiol)和11-羟基柳杉酚(11-hydroxyl sugiol), 再由SmCYP76AK1羟化11-羟基铁锈醇和11-羟基柳杉酚的C-20生成11, 20-二羟基铁锈醇(11, 20-hydroxy ferruginol)和11, 20-二羟基柳杉酚(11, 20-hydroxyl sugiol), 由于11, 20-二羟基柳杉酚不稳定, 可自氧化成10-羟甲基四氢丹参新酮(10-hydroxymerhyl tetrahydromiltirone)[13], 最后在其他CYP450氧化酶、脱羧酶、脱氢酶、还原酶的催化下形成丹参酮ⅡA、隐丹参酮等活性成分[14]。CYP450s是萜类骨架物质氧化修饰的主要酶类, 能将C-H键氧化, 使之成为可进一步转化的羟基。前期通过cDNA芯片[15]、基因组[2]和转录组[5]等筛选或预测到SmCYP450s 500余个, 其中33个在丹参根周皮显著高表达, 尤其是SmCYP76亚家族成员与丹参酮的下游合成途径关系密切[16]。目前, SmCYP76AH1SmCYP76AH3SmCYP76AK1已得到功能验证, 推测SmCYP71D375在丹参新酮合成的上游区域起作用, 分别从不同通路合成隐丹参酮和二氢丹参酮Ⅰ[17]

Figure 3 A schematic of the tanshinone biosynthetic pathway
1.2 丹酚酸类化合物的生物合成

丹酚酸类化合物属于酚酸类, 主要在丹参的韧皮部和木质部中合成和积累, 迄今已分离到30余种。从结构上看, 大多数丹酚酸类化合物可视为咖啡酸的衍生物, 如迷迭香酸(rosmarinic acid, RA)为咖啡酸与丹参素的二聚物, 丹酚酸B为迷迭香酸二聚体, 丹酚酸A由1分子丹参素与2分子咖啡酸缩合而成(图 2)[18]。因此, RA可能是大多数丹酚酸类化合物生物合成的共同前体。目前认为, 丹参中RA的合成有3种可能, 但均起源于苯丙烷代谢途径的两条平行支路。传统认为, RA由苯丙氨酸(L-phe)支路生成的4-香豆酰CoA (4-coumaroyl-CoA)和由酪氨酸(L-tyr)支路生成的4-羟基苯乳酸(4-hydroxyphenyllactic acid)在迷迭香酸合成酶(SmRAS)催化下生成2-氧-(4-香豆酰)-3-(4-羟基苯)-乳酸(4-coumaroyl-4'-hydroxyphenyllactic acid), 然后由细胞色素P450蛋白CYP98A14催化生成RA[19]。但Di等[20, 21]认为, 苯丙氨酸支路中4-香豆酸(4-coumaric acid)先被转化为咖啡酸(caffeic acid), 然后咖啡酰CoA (caffeoyl CoA)与4-羟基苯乳酸在SmRAS催化下生成咖啡酸-4-羟基苯乳酸(caffeoyl-4'-hydroxyphenyllactic acid), 再经CYP98A14转化成RA, 才是RA合成的主要路线。而酪氨酸支路的4-羟基苯乳酸经丹参素与4-香豆酰CoA在SmRAS催化下生成4-香豆酰-3', 4'-二羟基苯乳酸(4-coumaroyl-3', 4'-dihydroxyphenyllactic acid)后, 由CYP98A14催化形成RA是一条衍生路线(图 4)。相对于RA的合成, 丹酚酸下游生源途径还没有完全明确。Di等[21]、Li等[22, 23]认为丹酚酸B是由两分子RA在漆酶(lactase)等氧化酶的作用下生成苯环2位自由基和α位自由基, 然后二聚化反应直接生成或经丹酚酸E (salvianolic acid E)转化而成。然而, 综合Ag+和茉莉酸甲酯(MeJA)诱导后丹参毛状根中RA和丹酚酸B含量的变化情况[24], 过表达SmRAS仅能提高迷迭香酸的含量却无法提高丹酚酸B的含量, 且丹酚酸B的主要降解产物是丹参素, 并测得合成过程中间体—紫草酸(lithospermicacid)[25]; 以及MeJA和真菌提取物(YE)处理后酪氨酸支路上关键酶基因的响应与丹酚酸B合成的相关性等研究[26, 27]表明, 丹酚酸B也有可能由咖啡酸与RA结合后生成紫草酸, 然后由紫草酸与丹参素自发酯化而成(图 4)。

Figure 4 A schematic of the salvianolic acid biosynthetic pathway
2 丹参主要药效成分生物合成的转录调控研究

丹参酮和丹酚酸类化合物的生物合成受YE、Ag+、MeJA、水杨酸(SA)、脱落酸(ABA)、赤酶素(GA)和乙烯(Eth)等生物及非生物诱导子的诱导[1, 28], 其机制之一就是通过转录因子激活或抑制合成途径关键酶基因的表达以调控次生代谢产物的合成。

2.1 JAZ蛋白与bHLH转录因子家族

茉莉酸类物质(JAs)通过调控JA信号通路相应的转录因子进而调控丹参酮和丹酚酸类化合物的合成[29], 其介导的转录调控核心模型是SCFCOI1-JAZs-MYC2复合体。即正常生长的植物内源JAs水平较低, JAZ (jasmonate-ZIM domain)蛋白与MYC2等转录因子结合, 抑制茉莉酸早期响应基因的表达。当植物内源JAs或JA-异亮氨酸复合体(JA-Ile)含量增高时, JAZ蛋白与E3泛素连接酶SCFCOI1复合物相互作用, 并导致其自身被降解, 释放出MYC2等转录因子激活下游JAs应答基因的表达[30]

从丹参中分离的9条SmJAZ基因均受MeJA诱导, 过表达SmJAZs, 则SmRAS1SmCYP98A14等基因的表达量下降, 负调控丹酚酸B的合成。与丹酸酚不同的是, 仅SmJAZ3/4/8负调控丹参酮的合成, 而SmJAZ1/2/5/6/9则起正调控作用, 其中SmJAZ2/4/9通过调控SmCPS1SmCYP76AH1的表达来实现调控功能[31]。SmJAZs转录因子家族中, SmJAZ8的作用较为特殊, 因其缺失JAs结构域而无法与COI1蛋白结合, 主要通过N端的两性抑制基序(EAR)起稳定抑制转录因子的活性, 抑制SmCPS1SmCYP76AH1及处于SmCYP98A14下游酶基因的表达, 并参与JAs信号诱导的丹参初生代谢和次生代谢的负调控作用[31]。过表达SmJAZ8, 能显著抑制SmJAZ1/4/5/6/10 的表达, 甚至产生对JA不敏感的丹参表型, 而SmJAZ8-RNAi则提升家族其他成员的表达量[31], 是丹酚酸合成过程中较为重要的负调控因子。

SmJAZ作为JA信号通路中负调控茉莉酸信号从SCFCOI1复合体向下游应答基因传导的核心蛋白, 其启动子区域含有能与SmMYC2转录因子结合的元件[32, 33]。MYC2属于bHLH (basic helix-loop-helix transcription factors)转录因子家族, 含有高度保守的碱性螺旋-环-螺旋结构域, 其N端的碱性区域能识别DNA上的E-box和G-box位点, C端HLH结构域形成的二聚体或四聚体能与靶基因的启动子结合, 是调控JA信号通路基因表达的另一核心成员。研究表明:正常情况下, SmMYC2a/2b与JAZ1/2结合, 调控功能被抑制。当内源JAs或JA-Ile含量升高时, 释放出的SmMYC2a激活SmHCT6SmCYPA14; SmMYC2b激活SmCYPA14; SmMYC2激活SmTAT1SmPAL1SmC4HSmCYP98A14等基因的转录表达[34, 35], 正向调控丹酚酸的生物合成。但在已注释的131条SmbHLH转录因子中[36-38], 也有负调控丹酚酸合成的转录因子。如SmbHLH7能与JAZ3L结合, 负调控SmRAS1SmCYP98A14SmTAT1等基因表达[38]; SmbHLH37能与SmJAZ3/8结合, 负调控SmTAT1SmPAL1基因[39]; 而SmbHLH51与R2R3-MYB类转录因子AtPAP1形成调控转录复合体后, 正向调控丹酚酸类物质的合成(图 5)[40]

Figure 5 Transcription factor of SmbHLH regulation on salvianolic acid in JA signal pathway

除调控丹酚酸合成外, bHLH转录因子家族还对丹参酮的生物合成起调控作用。如SmMYC2可通过抑制SmbHLH74的转录, 削弱SmbHLH74对丹参酮合成关键酶基因SmHMGR1SmGGPPS1SmCYP76AH1的负调控[41]。JAMs在结构上比MYC2转录因子在N端少一个酸性激活结构域, 起负调控作用。拟南芥异源表达研究表明, SmJAM3能通过负调控AtMYC2/3/4和正调控AtJAM1以进一步调控MYC2下游靶基因AtERF1AtMYB75AtPDF1.2AtVSP2的表达。而在丹参中, SmJAM3、SmMYC2-likes分别与SmJAZ3、SmJAZ9蛋白互作, 上调表达SmGGPPS等基因的转录, 促进丹参酮合成[42]。此外, SmbHLH7能正调控SmHMGSSmGGPPS1SmCYP76AH1SmKSL1等丹参酮合成基因[38], SmbHLH10能与丹参酮合成关键酶SmDXS2SmCPS1SmCPS5基因启动子的G-box位点结合, 激活转录[43], 而SmbHLH148几乎能激活所有丹酚酸和丹参酮生物合成途径上的基因表达(图 6)[44]

Figure 6 Transcription factor of SmbHLH regulation on tanshinones in JA signal pathway

需要指出的是, JAZs作为一种阻遏蛋白, 通常起抑制作用[45]。但过表达SmJAZ1/2/5/6/9却显著提高转基因毛状根中丹参酮的含量, 且SmJAZs能与SmMYC2a、SmMYB39、SmMYC2b、SmPAP1等转录因子互作[32, 33], 促进SmMYC2aSmMYC2bSmPAP1表达, 抑制SmMYB39表达[33]。因此, SmJAZs蛋白在调控JA诱导的丹参次生代谢物合成中发挥中心枢纽作用, 存在复杂的共调控网络, 这种多个SmJAZs共同调控一种或一类次生代谢产物合成的机制还有待进一步阐明。此外, 由于采样或命名等原因而导致的不同研究者研究同一名称的转录因子(如SmbHLH7)却得到不一致结论的情况, 也应引起重视[38, 42]

2.2 MYB转录因子家族

MYB (v-myb avian myeloblastosis viral oncogene homolog)是植物中最大的转录因子家族, 根据所含串联重复序列(repeat, R)数量, 可将MYB转录因子分为4大类。其中, 参与植物次生代谢产物合成调控的转录因子主要来自R2R3-MYB亚家族, 可通过蛋白质互作、转录酶调控、氧化还原反应、ABA信号系统或参与JA信号系统行使功能。

Li等[46]从丹参全基因组中筛选出110个R2R3-MYB转录因子, 将其划分为37个亚族, 并预测S3、S4、S5、S6、S7、S13、S20、S21亚族对丹参酮和丹酚酸类化合物的合成具有调控作用。研究表明, S4亚族的R2R3-MYB转录因子主要通过乙烯响应因子相关的EAR对苯丙烷代谢产物的合成起抑制作用[47], 如SmMYB4通过抑制酚酸类上游苯丙烷代谢途径的SmPAL1SmC4H基因和丹参酮合成途径下游关键酶基因SmGGGPPS3, 负调控两类化合物的积累[48]; SmMYB39作为负调控因子, 除直接抑制迷迭香酸代谢途径关键酶基因SmTATSmC4H和丹参酮代谢途径SmDXS2SmDXRSmHMGR1SmGGPPSSmKSL1的转录外, 还能通过抑制SmbHLH7转录或干扰SmbHLH7与其他转录因子形成正调控MBW复合体等机制行使负调控功能[49, 50]。S6亚族转录因子SmPAP1与拟南芥中的PAP1蛋白的同源性高达75%, 在丹参中过表达拟南芥AtPAP1AtPAP2, 能不同程度正向调控苯丙烷类代谢途径上SmPALSmC4HSmRAS等多种酶基因的表达[51, 52]SmPAP1基因启动子区域上含有多种响应元件和bHLH结合位点[53], 可与SmMYC2转录因子协作, 共同激活迷迭香酸合成途径SmTATSmPAL1SmC4H等关键酶基因表达, 同时抑制丹参酮类生物合成关键酶基因SmCPS转录[54]。S7亚族的转录因子SmMYB111, 基因启动子区含有多种激素响应元件和MYB结合位点, 受MeJA、SA、GA和光等诱导, 能与SmTTG1、SMBHH51形成SmTTG1-SmMYB111-SmbHLH51三元转录复合物, 共同参与丹酚酸生物合成的正向调节[55]。SmMYB36位于S5和S15之间, 能与SmCPS1SmCYP76AH1SmKSL1等靶基因启动子区的MBS1、MBSⅠ、MBSⅡ响应元件及SmbHLH128发生互作, 促进丹参酮的积累和降低丹酚酸类物质的含量[56]。转录组和代谢组分析表明, SmMYB36对丹参酮类化合物合成的调控是全方位的, 苯丙烷途径、酪氨酸衍生途径、甲基赤藓糖醇磷酸途径和下游丹参酮生物合成途径的基因转录水平均发生了显著变化[57]

另外, S20亚族转录因子SmMYB98b、SmMYB9a和SmMYB9b, 均能激活MEP途径中SmDXS2SmDXRSmGGPPSSmKSL1等关键酶基因的转录, 显著提高丹参毛状根中丹参酮的含量[58, 59], 而SmMYB98则激活SmGGPPS1SmPAL1SmRAS1等基因的转录, 对丹参酮和丹酚酸的合成起正向调控作用[60]。而且, SmMYB98b和SmMYB9a还参与低磷对丹参酮的调控, 低磷诱导的丹参酮积累能被SmMYB98bSmMYB9a基因的过表达所抑制[61]。但这种调控方式有别于AtMYB2、AtMYB62对低磷的响应机制, AtMYB2、AtMYB62分别通过活化miR399的转录和调节GA信号及其代谢, 进而调节磷饥饿响应[62, 63], 而SmMYB98b、SmMYB9a则与低磷响应基因SmSPX2SmSPX4互作, 介导SPX对下游低磷响应基因(PSI)的调节作用(图 7)[61]

Figure 7 Transcription factor of SmMYB regulation on secondary metabolism in Salvia miltiorrhiza
2.3 WRKY转录因子家族

WRKY转录因子家族的DNA结合区包含1~2个WRKY核心结构域, C端为锌指结构, 主要通过ABA信号通路参与植物的生长发育、响应逆境胁迫以及参与次生代谢的调控, 也能与SA、JA等信号通路协同, 间接调控应答基因[64, 65]。丹参中共注释到WRKY转录因子69个, 其中从基因组数据中发现61个[66], 从转录组数据中筛选出8个[67]。Li等[66]对其中61条SmWRKYs基因进行研究, 发现42条能响应MeJA和Ag+胁迫, 推测SmWRKY3/9正向调控丹参酮的合成。Yu等[67]进一步推测SmWRKY19正向调控丹参酮、SmWRKY45/68正向调控丹酚酸、SmWRKY1/7/29/52/56/58对丹参酮和丹酚酸类化合物的生物合成均有正向调控作用。其中, Ⅲ类转录因子SmWRKY1, 已被证实能通过结合蛋白的方式对SmDXR进行正调控, 使转基因品系中丹参酮产量增加5倍[68]。Ⅰ类转录因子SmWRKY2, 能与SmCPS启动子区的W-box结合以激活转录, 正调控丹参酮的生物合成[69]。SmWRKY54[70]、SmWRKY3和SmWRKY70[71]等转录因子也能正向调控代谢途径上关键酶基因的转录, 促进丹参酮的合成。而受MeJA和YE强烈诱导的Ⅱa类转录因子SmWRKY9, 则正调控SmRAS1SmCYP98A14的转录(SmPAL3Sm4CL2Sm4CL3等酶基因可能也参与), 促进迷迭香酸的生物合成(图 8)[72]

Figure 8 Transcription factor of SmWRKY regulation on secondary metabolism in Salvia miltiorrhiza
2.4 EIL转录因子家族

JAs与乙烯都能促进植物次生代谢产物的积累, 两者在拟南芥中对基因的表达具有协同诱导、相互依赖作用, 任一激素信号通路被阻断, 对基因的诱导表达作用即消失。研究表明, EIN3 (ethyleneinsensitive factor 3)/EILs (EIN3-likes)转录因子位于乙烯与JA这两条信号通路的交叉点。正常情况下, 乙烯能促进EIN3/EILs的积累, 而JAZ蛋白可通过组蛋白去乙酰化酶(HDA6)直接抑制EIN3/EILs转录因子的活性, 当JAs与其受体SCFCOI1-JAZ结合后, JAZ蛋白随之降解, 从而解除HDA6对EIN3/EIL1的抑制, 启动EIN3/EIL1调控的基因表达[73]。丹参SmEIN3转录因子响应ABA、YE、MeJA和SA的诱导, 但GA抑制其表达。在转录水平上丹参SmEIN3不响应乙烯诱导, 乙烯调节的是转录后水平的EIN3/EIL蛋白及其活性, 以激活丹酚酸合成关键酶基因Sm4CL1SmCYP98A14SmHPPRSmRAS和丹参酮合成关键酶基因SmDXSⅡSmDXRSmCYP76AH1SmKSLSmGGPPSSmHMGSSmAACTSmHMGRSmCPS的转录。从SmEIN3与SmMYC2、SmJAZs存在互作现象分析, 丹参酮和丹酚酸类化合物的生物合成调节中可能存在乙烯信号通路与JA信号通路的交流, 两者起共同调控作用(图 9)[74]

Figure 9 Transcription factor of SmEIN3/EILs, SmDELLA and SmERF regulation on secondary metabolism in Salvia miltiorrhiza
2.5 GRAS转录因子家族

GA3处理后, 能激活SmCPS1SmKSL1SmPALSmTAT等关键酶基因的表达, 促进丹参酮和丹参酚类物质的合成[75]。DELLA是GRAS转录因子超家族中的重要成员, 其N端具有GA受体GID1结合域, 为GA信号途径的负调控因子。GA可通过调控SLEEPY1等蛋白以促进DELLA降解, 而DELLA的降解则促进JAs的合成与JAZ降解, 从而进一步促进了DELLA的去抑制化[76]。同时, DELLA蛋白也可通过结合不同的转录因子介导GA信号调控下游靶基因[77], 如以DELLA-JAZ蛋白互作的形式达到GA-JA途径调控网络的实现[78]; 在缺氮时, DELLA通过与PAP1蛋白相互作用, 增强PAP1对靶基因的调控(图 9)[79]。Bai等[80]从丹参中克隆出5个能响应GA或乙烯诱导的SmGRAS转录因子, 通过激活SmCPS1SmKSL1的转录, 正向调控丹参次生代谢的合成。Wang[81]从丹参中筛选出同时响应GA3、水杨酸(SA)和JAs信号途径的SmGRAS转录因子5个, 并鉴定出11个亚家族共34个SmGRAS转录因子, 其中4个转录因子属于SmDELLA亚家族, 都能与SmJAZ1、SmJAZ3、SmMYB36、SmMYC2等蛋白互作, 推测对丹参次生代谢产物的合成具有调控作用。

2.6 ERF类转录因子家族

ERF类转录因子属于AP2/ERF家族成员, 在AP2结构域的N-端具有特异识别顺式作用元件的β-折叠结构, 而C-端的α-螺旋结构则用于与其他转录因子或DNA的GCC-box相互作用。AP2/ERF类转录因子共有5个亚族, ERF (ethylene response factors)亚族种类最多, 大多数成员响应乙烯信号通路(也可响应JA、SA信号通路), 在植物生长、发育和次生代谢中扮演重要作用[82]。Ji等[83]从丹参基因组数据中注释到79个ERF转录因子, 并根据丹参酮的分布、转录因子的表达模式及启动子顺式元件等推测SmERF128/152与丹参酮生物合成相关, SmERF8/166与丹酚酸合成相关。SmERF2和SmO3L3通过结合(A/G/C) GCCGCC型的GCC-box, 分别激活丹参酮合成途径的SmDXRSmDXS2SmHMGSSmKSL和丹酚酸合成途径的Sm4CL1SmRAS等酶基因表达[84]。SmERF6和SmERF8可识别SmCPS1SmKSL1启动子区的乙烯反应元件GCC-box, 正向调控丹参酮的生物合成[85, 86]。SmERF115受MeJA诱导, 但不受YE、SA、ET诱导, 能直接结合SmRAS1启动子激活表达(可能也激活SmPAL3SmTAT3Sm4CL5等), 促进丹酚酸类化合物的生成[87]。而SmERF1L1则对MeJA、YE、SA、ET的诱导均有响应, 能激活SmDXRSmDXS2SmHMGSSmKSL的转录, 促进丹参酮的合成[88]。SmERF128可结合靶基因的GCC盒、CRTDREHBCF2 (CBF2)基序和RAV1AAT (RAA)基序, 激活SmCPS1SmKSL1SmCYP76AH1的表达(图 9)[89]

2.7 LBD转录因子家族

LBD (lateral organ boundaries domain)是一类植物特有的转录因子家族。具有完整亮氨酸拉链基序(Class Ⅰ)的成员, 主要参与组织发育的调控, 而不具有完整亮氨酸拉链基序的成员(Class Ⅱ), 主要与花青素生物合成、氮代谢的调控相关。从丹参基因组中鉴定出51个SmLBD, 90%的SmLBD具有保守的CX2CX6CX3C结构, 74%的SmLBD在叶片表达, 推测SmLBD9/13/21/50等与丹酚酸化合物的合成关系密切, SmLBD44与丹参酮的合成调控相关[90]。研究表明, LBD转录因子的保守区段即LOB结构域可与bHLH、MYB转录因子相互作用, 参与JA信号下游途径调控[91], 且同类LBD转录因子的功能相对保守, 但作用方式有所区别[92]。如SmLBD16/23/50均能负向调控丹酚酸的合成, 且与SmJAZ1、SmMYB36/97、SmbHLH37和SmMYC2a/2b等转录因子存在蛋白互作, 但SmLBD16在调控苯丙烷途径关键酶基因表达时需SmPAP1参与, 而SmLBD23则不需要[93]

3 总结与展望

近年来, 丹参酮和丹酚酸类化合物的生物合成及其转录因子调控机制研究取得长足进展, 一方面得益于丹参全基因组图谱的完成, 不同组织、器官的转录组测序, 使其能在基因组和转录组层面整体分析与丹参酮和丹酚酸类化合物生物合成相关的差异基因和转录因子[94], 另一方面得益于拟南芥、烟草、水稻等模式植物研究中所建立的高效成熟的转化系统。但是, 丹参作为药用模式植物, 其次生代谢产物的生物合成及调控机制研究, 还需继续深入, 主要体现在4个方面: ①丹参酮和丹酚酸类化合物的生物合成途径还未被完全阐明, 急需明确参与丹参酮后期修饰及迷迭香酸下游途径的关键酶, 并进一步揭示其合成、转运、调节、修饰的新基因及作用机制。②丹参中转录因子众多, 家族成员庞大, 同一家族(亚家族)成员功能相似等, 大大增加了转录因子调控机制研究的难度, 但是家族成员之间及不同家族转录因子之间的相互作用、响应的信号通路、参与的合成途径等错综复杂的调控关系, 仍是今后的研究重点。③部分转录因子的研究, 如SPL (SQUAMOSA promoter binding protein-like)转录因子[95]、bZIP (basic region/leucine zipper motif)转录因子[96], 还停留在功能预测层面, 急需进一步深入。同时, 转录因子自身的转录也受到其他基因的调控, 如microRNAs、ncRNAs等[97], 但这方面的研究较少。④人工设计并构建新的、具有特定生理功能的生物系统, 是一种极具潜力的丹参活性成分资源获取方法。如通过构建SmHMGR催化活性域-SmGGPPS, SmFPS融合蛋白-SmKSL, SmCPS融合蛋白酵母工程菌, 使次丹参酮二烯的产量达到了365 mg·L-1 [98]; 组合改造FPP合成酶和GGPP合成酶, 将次丹参酮二烯的产量提高到488 mg·L-1 [99]; 引入CYP76AH1SmCPR1基因, 将铁锈醇的最高产量提高到10.5 mg·L-1 [12]。而转录因子能同时诱导一个或多个基因的协同表达, 调控效果优于单纯提高单个或多个结构基因的表达, 是另一大幅提高合成酶基因表达及活性物质产量的可能方法, 值得进一步深入研究。

作者贡献:詹忠根主要负责选题, 文献检索和初稿写作, 李杏主要负责文稿修改与校对。

利益冲突:无利益冲突。

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