丹参迷迭香酸合成途径相关基因的功能研究
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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。
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.
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。
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.
引文
[1]国家药典委员会.中华人民共和国药典,一部[M].北京：化学工业出版社,2005：73.
[2]Zhou L, Zuo Z, Chow MS. Danshen:an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use [J]. J. Clin. Pharmacol,2005,45:1345-1359.
[3]Ryu SY, Ochlee C, Choi S, et al. In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza [J]. Planta Med,1997,63(4):339-342.
[4]Jang SI, Jeong SI, Kim KJ, et al. Tanshinone IIA from Salvia miltiorrhiza inhibits inducible nitric oxides ynthase expression and production of TNF-alpha, IL-lbetaand IL-6 in activated RAW 264.7 cells [J]. Planta Med,2003,69: 1057-1059.
[5]Hase K, Kasimu R, Basnet P, et al. Preventive effect of lithospermate B from Salvia miltiorrhiza on experimental hepatitis induced by carbon tetrachloride or D-galactosamine/lipopolysaccharide [J]. Planta Med,1997,63:22-26.
[6]Tanaka T, Morimoto S, Nonaka G, et al. Magnesium and ammonium-potassium lithospermates B, the active principles having a uremia-preventive effect from Salvia miltiorrhiza [J]. Chem. Pharm. Bull,1989,37:340-344.
[7]Arda N, Goren N, Kuru A, et al. Saniculoside N from Sanicula europaea [J]. J. Nat. Prod,1997,60:1170-1173.
[8]赵红霞,张利,凡星,等.丹参、黄花鼠尾和雪山鼠尾染色体数目的研究[J].中国中药杂志,2006,31：1847-1849.
[9]张晓媛,王彩红.利用根尖染色体鉴别药用植物丹参、甘西鼠尾的种子[J].安徽农业科学,2008,36(21)：9155-9156.
[10]Yan YP, Wang ZZ. Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method [J]. Plant Cell Tissue Organ Cult,2007,88:175-184.
[11]刘文婷,梁宗锁,蒋传中,等.丹参生殖生物学特性研究[J].现代中药研究与实践,2004,18(5)：17-20.
[12]Liu AH, Lin YH, Yang M, et al. High-performance liquid chromatographic determination of tanshinones in the roots of Salvia miltiorrhiza and related traditional Chinese medicinal preparations [J]. J. Pharm. Sci,2006,9(1):1-9.
[13]Wang X, Morris-Natschke SL, Lee KH. New developments in the chemistry and biology of the bioactive constituents of danshen [J]. Med. Res. Rev,2007,27 (1): 133-148.
[14]黄璐琦,戴住波,吕冬梅,等.探讨道地药材研究的模式生物及模型[J].中国中药杂志,2009,34(9)：1063-1066.
[15]王庆浩,陈爱华,张伯礼.丹参：一种中药研究的模式生物[J].中医药学报,2009,37(4)：1-4.
[16]Wang X, Geng Y, Li F, et al. Large-scale separation of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by pH-zone-refining countercurrent chromatography [J]. J. Sep. Sci,2007,30:3214-3217.
[17]Ma L J, Zhang XZ, Guo H, et al. Determination of four water-soluble compounds in Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometric electrode array system [J]. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci,2006,833:260-263.
[18]Lam FF, Yeung JH, Kwan YW, et al. Salvianolic acid B, an aqueous component of Danshen (Salvia miltiorrhiza), relaxes rat coronary artery by inhibition of calcium channels [J]. Eur. J. Pharmacol,2006,553:240-245.
[19]林汉钦.从丹参分离出一种新丹参酮成分[J].中华药学杂志,1993,45(9)：615-618.
[20]Choi J S, Kang H S, Jung H A, et al. A new cyclic phenyllactamide from Salvia miltiorrhiza [J]. Fitoterapia,2001,72(1):30-34.
[21]Zhou L, Chow M, Zuo Z. Improved quality control method for Danshen products-consideration of both hydrophilic and lipophilic active components [J]. J. Pharm. Biomed. Anal,2006,41(3):744-750.
[22]Liu YR, Qu SX, Maitz MF, et al. The effect of the major components of Salvia miltiorrhiza Bunge on bone marrow cells [J]. J. Ethnopharmacol,2007, 111(3):573-83.
[23]刘陶世,黄耀洲.丹参成分、制剂和质量控制研究概况[J].南京中医药大学学报,1998,14(4)：255-256.
[24]郭济贤.丹参的研究与临床应用[M].北京：中国医药科技出版社,1992：29.
[25]Li H C, Chang W L. Diterpenoids from Salvia miltiorrhiza [J]. Phytochemistry, 2000,53(8):951-953.
[26]Liu AH, Li L, Xu M, et al. Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by HPLC-DAD method [J]. J. Pharm. Biomed. Anal,2006,41:48-56.
[27]Zhang H, Yu C, Jia JY, et al. Contents of four active components in different commercial crude drugs and preparations of Danshen (Salvia miltiorrhiza) [J]. Acta. Pharmaco. Sin,2002,23:1163-1168.
[28]杜冠华,张均田.丹酚酸A对小鼠脑缺血再灌注致学习记忆功能障碍的改善作用及作用机制[J].药学学报,1995,30(3)：184.
[29]Li YG, Song L, Liu M, et al. Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma (Danshen) [J]. J. Chromatogr. A,2009,1216:1941-1953.
[30]Tsai MK, Lin YL, Huang YT. Effects of salvianolic acids on oxidative stress and hepatic fibrosis in rats [J]. Toxicol Appl Pharmacol,2010,242:155-164.
[31]Zhao GR, Zhang HM, Ye TX, et al. Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B[J]. Food Chem Toxicol, 2008,46:73-81.
[32]冯玲玲,周吉源.丹参的研究现状与应用前景[J].中国野生植物资源,2004,23(2)：4-7.
[33]陈震.丹参生长与隐丹参酮含量的关系[J].中药通报,1983,8(1)：2.
[34]文家富,王刚云,陈光华,等.商洛市丹参主要病虫害调查及综合防治技术[J].陕西农业科学,2009,1：210-211.
[35]Yuan JM, Tao LL, Xu JT. Immobilization of callus tissue cells of Salvia miltiorrhiza and the characteristics of their products [J]. Chin. J. Biotechnol,1990, 6(3):199-205.
[36]王康才,罗庆云,陈红霞,等.丹参愈伤组织中次生代谢产物形成的研究[J].中国中药杂志,1998,23(10)：592-594,638.
[37]Huang L, Liu D, Hu Z. Effects of phytohormones on growth and content of depsides in Salvia miltiorrhiza suspension cells [J]. Zhong Yao Cai (in Chinese), 2000,23(1):1-4.
[38]Wu JY, Ng J, Shi M, et al. Enhanced secondary metabolite (tanshinone) production of Salvia miltiorrhiza hairy roots in a novel root-bacteria coculture process. Appl Microbial Biotechnol,2007,77 (3):543-550.
[39]Shimomura K, Kitazawa T, Okamura N,et al. Tanshinone production in adventitious roots and regenerates of Salvia miltiorrhiza[J]. J. Nat. Prod,1991, 54(6):1583-1587.
[40]黄链栋,胡之壁,刘涤.丹参发状根再生植株的研究[J].上海中医药杂志,1996，10：40.
[41]张荫麟,宋经元,祁建军,等.农杆菌转化后丹参植株再生[J].中国中药杂志,1997,22(5)：274-276.
[42]宋经元,张荫麟,祁建军,等.丹参冠瘿组织丹参高产株系选择和丹参酮的产生[J].生物工程学报,1997,13(3)：317-319.
[43]Wang XY, Cui GH, Huang LQ, et al. Effects of methyl jasmonat on accumulation and release of tanshinones in suspension cultures of Salvia miltiorrhiza hairy root[J].中国中药杂志,2007,32(4)：300-302.
[44]Yan Q, Shi M, Ng J, et al. Elicitor-induced rosmarinic acid accumulation and secondary metabolism enzyme activities in Salvia miltiorrhiza hairy roots [J]. Plant Sci.2006,170:853-858.
[45]何水林,郑金贵,王晓峰,等.植物次生代谢：功能、调控及其基因工程[J].应用与环境生物学报,2002,8(5)：558-563.
[46]Yan YP, Wang ZZ, Tian W, et al. Generation and analysis of expressed sequence tags from the medicinal plant Salvia miltiorrhiza [J]. Sci. China Life Sci,2010,53: 273-285.
[47]崔光红,黄璐琦,邱德有,等.丹参功能基因组学研究Ⅱ—丹参毛状根不同时期基因表达谱分析[J].中国中药杂志,2007,32(13)：1267-1272.
[48]崔光红.丹参道地药材cDNA芯片构建及毛状根基因表达谱研究[D].北京：中国中医科学院,2006.
[49]Song J, Wang Z. Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmP4L1) from Salvia miltiorrhiza [J]. Mol. Biol. Rep,2009,36:939-52.
[50]刘世海.丹参肉桂酸4-羟化酶基因克隆及功能研究[D].陕西：陕西师范大学,2007.
[51]Huang B, Duan Y, Yi B, et al. Characterization and expression profiling of cinnamate 4-hydroxylase gene from Salvia miltiorrhiza in rosmarinic acid biosynthesis pathway[J]. Russ. J. Plant Physiol,2008,5:431-440.
[52]Zhao SJ, Hu ZB, Liu D, et al. Two divergent members of 4-coumarate:coenzyme a ligase from Salvia miltiorrhiza Bunge:cDNA cloning and functional study [J]. J. Integr. Plant Biol,2006,48:1355-1364.
[53]Huang B, Yi B, Duan Y, et al. Characterization and expression profiling of tyrosine aminotransferase gene from Salvia miltiorrhiza (Dan-shen) in rosmarinic acid biosynthesis pathway [J]. Mol. Biol. Rep,2008,35:601-612.
[54]段艳冰.丹参迷迭香酸代谢酪氨酸支路重要基因克隆及调控分析[D].上海：第二军医大学,2006.
[55]Xiao Y, Di P, Chen J, et al. Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (Smhppd) from Salvia miltiorrhiza hairy root cultures [J]. Mol. Biol. Rep,2009,36:2019-2029.
[56]Liao P, Zhou W, Zhang L, et al. Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Salvia miltiorrhiza [J]. Acta. Physiol. Plant,2009,31(3):565-572.
[57]化文平.丹参GGPP合酶基因的克隆及表达分析[D].陕西：陕西师范大学,2008.
[58]刘丛玲,王喆之.丹参晚期胚胎蛋白基因SmLEA的克隆及表达分析[J].生物技术通报,2009,5：80-84.
[59]刘世海,闫亚平,王喆之.丹参DHN2基因的克隆与序列分析[J].分子植物育种,2005,3(1)：66-70.
[60]王铭楷,闫亚平,田薇,等.丹参DHN1基因的克隆与序列分析[J].西北植物学报,2004,24(11)：1990-1995.
[61]张成,王喆之.丹参钙调蛋白cDNA的克隆及其反义表达载体的构建[J].分子植物育种,2006,4(3)：339-344.
[62]韩立敏.丹参APX和GPX基因克隆及其表达分析[D].陕西：陕西师范大学,2007.
[63]Tanaka T, Morimoto S, Nonaka G, et al. Magnesium and ammonium-potassium lithospermates B, the active principles having a uremia-preventive effect from Salvia miltiorrhiza[J]. Chem. Pharm. Bull,1989,37:340-344.
[64]Petersena M, Simmonds MS. Rosmarinic acid [J]. Phytochemistry,2003, 62:121-125.
[65]Petersen M, Ha'usler E, Karwatzki B, et al. Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth [J]. Planta,1993,189:10-14.
[66]Petersen M. Current status of metabolic phytochemistry[J]. Phytochemistry 2007; 68:2847-60.
[67]Grotewold E. Transcription factors for predictive plant metabolic engineering:are we there yet[J]. Curr. Opin. Biotechnol,2008,19:138-144.
[68]Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana [J]. Curr. Opin. Plant. Biol,2001,4:447-456.
[69]Wilkins O, Nahal H, Foong J, et al. Expansion and diversification of the populus R2R3-MYB family of transcription factors [J]. Plant Physiol,2009,149:981-993.
[70]Hemm MR, Herrmann KM, Chapple C. AtMYB4:a transcription factor general in the battle against UV [J]. Trends Plant Sci,2001,6:135-136.
[71]Preston J, Wheeler J, Heazlewood J, et al. AtMYB32 is required for normal pollen development in Arabidopsis thaliana [J]. Plant J,2004,40:979-995.
[72]Borevitz J O, Xia Y J, Blount J, et al. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis [J]. Plant Cell,2000,12: 2383-2393.
[73]Galis I, Simek P, Narisawa T, et al. A novel R2R3 MYB transcription factor NtMYBJSl is a methyl jasmonate-dependent regulator of phenylpropanoid conjugate biosynthesis in tobacco [J]. Plant J,2006,46:573-592.
[74]Ritter H, Schulz GE. Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase [J]. Plant Cell,2004,16: 3426-3436.
[75]Lois R, Dietrich A, Hahlbrock K, et al. A phenylalanine ammonia-lyase gene from parsley:structure, regulation and identification of elicitor and light responsive cis-acting elements [J]. EMBO J,1989,8:1641-1648.
[76]Dixon RA, Paiva NL. Stress-induced phenylpropanoid metabolism [J]. Plant Cell, 1995,7:1085-1097.
[77]MacDonald MJ, D'Cunha GB. A modern view of phenylalanine ammonia lyase [J]. Biochem Cell Biol,2007,85:273-282.
[78]Shadle GL, Wesley SV, Korth KL, et al. Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase [J]. Phytochemistry,2003,64:153-161.
[79]Ohl S, Hedrick SA, Chory J, et al. Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis [J]. Plant Cell,1990,2:837-848.
[80]Yeo YS, Lee SW, Kim YH, et al. Restriction mapping of phenylalanine ammonia-lyase gene family in tomato(Lycopersicon esculentum) [J]. RDA J. Agric. Sci. Biotechnol,1994,36(2):187-192.
[81]Pellegrini L, Rohfritsch O, Fritig B, et al. Phenylalanine ammonia-lyase in tobacco: molecular cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the response to a fungal elicitor [J]. Plant Physiol,1994, 106:877-886.
[82]Elkind Y, Edwards R, Mavandad M, et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene [J]. Proc. Nadl. Acad. Sci,1990, 81:9057-9061.
[83]Chen F, Reddy MS, Temple S, et al. Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wallbound ferulic acid in alfalfa (Medicago sativa L.) [J]. Plant J,2006,48:113-124.
[84]Rohde A, Morreel K, Ralph J, et al. Molecular phenotyping of the pall and pal2 mutants of Arabidopsis Thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism [J]. Plant Cell,2004, 16:2749-2771.
[85]Mahesh V, Rakotomalala JJ, Gal LL, et al. Isolation and genetic mapping of a Coffea canephora phenylalanine ammonia-lyase gene (CcPAL1) and its involvement in the accumulation of caffeoyl quinic acids [J]. Plant Cell Rep,2006, 25(9):986-992.
[86]Liu R, Xu S, Li J, et al. Expression profile of a PAL gene from Astragalus membranaceus var. mongholicus and its crucial role in flux into flavonoid biosynthesis [J]. Plant Cell Rep,2006,25(7):705-710.
[87]Razzaque A, Ellis BE. Rosmarinic acid production in Coleus cell cultures [J]. Planta,1977,137:287-291.
[88]Chen H, Chen F. Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures [J]. Plant Cell Rep,2000, 19:710-717.
[89]Morant M, Bak S, Moller BL, et al. Plant cytochromes P450:tools for pharmacology, plant protection and phytoremediation [J]. Curr Opin Biotechnol, 2003,14(2):151-162.
[90]Chen AH, Chai YR, Li JN, et al. Molecular cloning of two genes encoding cinnamate 4-hydroxylase (c4h) from oilseed rape (Brassica napus) [J]. J. Biochem. Mol. Biol,2007,40:247-260.
[91]Blount JW, Korth KL, Masoud SA, et al. Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway [J]. Plant Physiol,2000,122: 107-116.
[92]Ro DK, Mah N, Ellis BE, et al. Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase [J]. Plant Physiol,2001,126(1):317-329.
[93]Koopmann E, Logemann E, Hahlbrock K. Regulation and functional expression of cinnamate 4-hydroxylase from parsley [J]. Plant Physiol,1999,119(1):49-56.
[94]Teutsch HG, Hasenfratz MP, Lesot A, et al. Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway [J]. Proc. Natl. Acad. Sci. USA,1993,90(9):4102-4106.
[95]Ranjeva R, Boudet A, Faggiox R. Phenolic metabolism in petunia tissues. IV. Properties of p-coumarate:coenzyme a ligase isoenzymes [J]. Biochimie,1976,58: 1255-1262.
[96]Ehlting J, Buttner D, Wang Q, et al. Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms [J]. Plant J,1999,19:9-20.
[97]Hu WJ, Kawaoka A, Tsai CJ, et al. Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides) [J]. Proc. Natl. Acad. Sci. USA,1998,95,5407-5412.
[98]Kumer A, Ellis BE.4-Coumarate:CoA ligase gene family in Rubus idaeus:cDNA structures, evolution, and expression [J]. Plant Mol. Biol,2003,31:327-340.
[99]Hamberger B, Hahlbrock K. The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapateactivating and three commonly occurring isoenzymes [J]. Proc. Nadl. Acad. Sci,2004,101:2209-2214.
[100]De Eknamkul W, Ellis BE. Tyrosine aminotransferase:the entrypoint enzyme of the tyrosine-derived pathway in rosmarinic acid biosynthesis [J]. Phytochemistry, 1987,26:1941-1946.
[101]Heike HC, Janine G, Iris S, et al. Tocopherol content and activities of tyrosine aminotransferase and cystine lyase in Arabidopsis under stress conditions [J]. J. Plant Physiol,2005,162:767-770.
[102]Petersen M, Alfermann AW. Two new enzymes of rosmarinic acid biosynthesis from cell cultures of Coleus blumei:hydroxyphenylpyruvate reductase and rosmarinic acid synthase [J]. Z Naturforsch,1988,43c:501-504.
[103]Kim KH, Janiak V, Petersen M. Purification,cloning and functional expression of hydroxyphenylpyruvate reductase involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei [J]. Plant Mol Biol,2004,54:311-323.
[104]肖莹.丹参酚酸类成分生源合成的调控研究[D].上海：第二军医大学,2009.
[105]Berger A, Meinhard J, Petersen M. Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases [J]. Planta,2006,224:1503-1510.
[106]St. Pierre B, De Luca V. Evolution of acyltransferase genes:origin and diversification of the BAHD superfamily of acyltransferases involved in secondary metabolism [J]. Rec. Adv. Phytochem,2000,34:285-316.
[107]D'Auria JC. Acyltransferases in plants:a good time to be BAHD [J]. Curr. Opin. Plant. Biol,2006,9:331-340.
[108]St. Pierre B, LaXamme P, Alarco AM, et al. The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A dependent acyl transfer [J]. Plant J,1998,14:703-713.
[109]Dudareva N, D'Auria JC, Nam KH, et al. Acetyl-CoA:benzylalcohol acetyltransferase — an enzyme involved in floral scent production in Clarkia breweri [J]. Plant J,1998,14:297-304.
[110]Hoffmann L, Maury S, Martz F, et al. Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism [J]. J. Biol. Chem,2003,278:95-103.
[111]Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcine synthase gene into Petunia results in reversible co-suppression of homologous genes in trans [J]. Plant Cell,1990,2:279-289.
[112]Fire A, Xu SQ, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature,1998, 39(1):806-811.
[113]何承伟,刘芳,刘新光,等.RNA干涉作用机制的研究进展[J].医学综述,2005,11(4)：291-294.
[114]徐秉良,师桂英,王延秀.RNA干扰与基因敲除[J].生物技术通报,2004,15(4)：386-388.
[115]孙建国,廖荣霞,陈正堂.RNA干涉分子机制研究进展[J].生物化学与生物 物理进展,2002,29(5)：678-680.
[116]周小云,陈信波,向建华.RNA技术及在植物功能基因组研究中的应用[J].生物学杂志,2005,22(2)：38-41.
[117]Bezanilla M, Pan A, Ralph SQ. RNA interference in the moss physcomitrella patens [J]. Plant physiol,2003,133(2):470-474.
[118]Shinjiro O, Hirotaka U, Yube Y, et al. Producing decaffeinated coffee plants [J]. Nature,2003,423(19):823.
[119]Gianfranco D, Raffaela T, Ralf W, et al. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase [J]. BMC Plant Biol,2006,6:13.
[120]Xiong AS, Yao QH, Peng RH, et al. Diferent effection ACC oxidase gene silencing triggered by RNA interference in transgenic tomato [J]. Plant Cell Rep, 2005,23:639-646.
[121]Coleman HD, Park J, Nair R, et al. RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism [J]. Proc. Nadl. Acad. Sci,2008,105:4501-4506.
[122]栾雨时,包永明.生物工程实验技术手册[M].北京：化学工业出版社,2005：167-168.
[123]Baxevanis A D, Ouellette B F F(著),李衍达,孙之荣等(译).生物信息学[M].北京：清华大学出版社,2000：231-250.
[124]Mount D W(著),钟扬,王莉,等.生物信息学[M].北京：高等教育出版社,2003：301-345.
[125]Kyte J, Doolittler F. A simple method for displaying the hydropathic character of a protein [J]. J. Mol. Biol,1982,157 (6):105-132.
[126]Nielsen H, Engelbrecht J, Brunak S, et al. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites [J]. Protein Eng, 1997,10 (1):1-6.
[127]Bendtsen J D, Nielsen H, Heijne G V, et al. Prediction of signal peptides:Signal P 3.0 [J]. J. Mol. Biol,2004,340:783-795.
[128]翟中和,王喜忠,丁明孝.细胞生物学[M].北京：高等教育出版社,2000：323-328.
[129]王镜岩,朱圣庚,徐长法(主编).生物化学(上册)(第三版)[M].北京：高等教育出版社,2002：157-181.
[130]Geourjon C, Deleage G. SOPMA:Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments [J]. Comput. Appl. Biosci,1995,11 (6):681-684.
[131]Nakazawa A, Nozue M, Yasuda H, et al. Expression pattern and gene structure of phenylalanine ammonia-lyase in Pharbitis nil [J]. J. Plant Res,2001,114:323-328.
[132]Logemann E, Parniske M, Hahlbrock K. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley [J]. Proc. Natl. Acad. Sci. USA,1995,92:5905-5909.
[133]Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture [J]. Physiol Plant,1962,15:473-497.
[134]闫亚平.基于EST技术的丹参大规模基因克隆及其外源基因转化技术研究[D].陕西：陕西师范大学,2005.
[135]Wesley SV, Helliwell CA, Smith NA, et al. Construct design for efficient, effective and high throughput gene silencing in plants [J]. Plant J,2001,27: 581-590.
[136]Gleave AP. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome [J]. Plant Mol Biol,1992,20:1203-1207.
[137]Lee HS, Wicker L. Anthocyanin pigments in the skin of lychee fruit [J]. J. Food Sci,1991,56(2):466-468,483.
[138]Dewanto V, Wu X, Adorn KK, et al. Thermal proeessing enhances the nutritional value of tomatoes by inereasing total antioxidant activity [J]. J. Agric. Food Chem, 2002,50:3010-3014.
[139]Brand-Williams W, Cuvelier ME, Berset C. Use of free radical method to evaluate antioxidant activity [J]. Lebensm. Wiss. Technol,1995,28:25-30.
[140]Dorman H, Peltoketoa A, Hiltunena R, et al. Characterisation of the antioxidant properties of de-odourised aqueous extracts from selected Lamiaceae herbs [J]. Food Chemistry,2003,83:255-262.
[141]Jin H, Cominelli E, Bailey P, et al. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis [J]. EMBO J,2000, 19:6150-6161.
[142]Petersen M, Abdullah Y, Benner J, et al. Evolution of rosmarinic acid biosynthesis [J]. Phytochemistry,2009,70:1663-1679.
[143]Nakayama T, Suzuki H, Nishino T. Anthocyanin acyltransferases:specificities, mechanism, phylogenetics, and applications [J]. J. Mol. Catal., B Enzym,2003,23: 117-132.
[144]Tacke E, Korfhage C, Michel D, et al. Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm [J]. Plant J,1995,8:907-917.
[145]Negruk V, Yang P, Subramanian M, et al. Molecular cloning and characterization of the CER2 gene of Arabidopsis thaliana [J]. Plant J,1996,9:137-145.
[146]Xia Y, Nikolau BJ, Schnable PS. Cloning and characterization of CER2, an Arabidopsis gene that affects cuticular wax accumulation [J]. Plant Cell,1996,8: 1291-1304.
[147]Bayer A, Ma XY, Stockigt J. Acetyltransfer in natural product biosynthesis— functional cloning and molecular analysis of vinorine synthase [J]. Bioorg Med. Chem,2004,12:2787-2795.
[148]Shalit M, Guterman I, Volpin H, et al. Volatile ester formation in roses. Identification of an acetyl-coenzyme A geraniol/citronellol acetyltransferase in developing rose petals [J]. Plant Physiol,2003,131:1868-1876.
[149]Boatright J, Negre F, Chen XL, et al. Understanding in vivo benzenoid metabolism in Petunia petal tissue [J]. Plant Physiol,2004,135:1993-2011.
[150]Wang J, De Luca V. The biosynthesis and regulation of biosynthesis of Concord grape fruit esters, including'foxy'methyl anthranilate [J]. Plant J,2005, 44:606-619.
[151]Wagner A, Ralph J, Akiyama T, et al. Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiate [J]. Proc. Natl. Acad. Sci. USA,2007,104:11856-11861
[152]Comino C, Hehn A, Moglia A, et al. The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway [J]. BMC Plant Biol,2009,9:30.
[153]Rischer H, Oresic M, Seppanen-Laakso T, et al. Gene-tometabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells [J]. Proc. Natl. Acad. Sci. USA,2006,103:5614-5619.
[154]Zulak KG, Cornish A, Daskalchuk TE, et al. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism [J]. Planta,2007,225:1085-1106.
[155]Xiao Y, Gao S, Di P, et al. Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures [J]. Physiol. Plant,2009,137:1-9.
[2]Zhou L, Zuo Z, Chow MS. Danshen:an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use [J]. J. Clin. Pharmacol,2005,45:1345-1359.
[3]Ryu SY, Ochlee C, Choi S, et al. In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza [J]. Planta Med,1997,63(4):339-342.
[4]Jang SI, Jeong SI, Kim KJ, et al. Tanshinone IIA from Salvia miltiorrhiza inhibits inducible nitric oxides ynthase expression and production of TNF-alpha, IL-lbetaand IL-6 in activated RAW 264.7 cells [J]. Planta Med,2003,69: 1057-1059.
[5]Hase K, Kasimu R, Basnet P, et al. Preventive effect of lithospermate B from Salvia miltiorrhiza on experimental hepatitis induced by carbon tetrachloride or D-galactosamine/lipopolysaccharide [J]. Planta Med,1997,63:22-26.
[6]Tanaka T, Morimoto S, Nonaka G, et al. Magnesium and ammonium-potassium lithospermates B, the active principles having a uremia-preventive effect from Salvia miltiorrhiza [J]. Chem. Pharm. Bull,1989,37:340-344.
[7]Arda N, Goren N, Kuru A, et al. Saniculoside N from Sanicula europaea [J]. J. Nat. Prod,1997,60:1170-1173.
[8]赵红霞,张利,凡星,等.丹参、黄花鼠尾和雪山鼠尾染色体数目的研究[J].中国中药杂志,2006,31：1847-1849.
[9]张晓媛,王彩红.利用根尖染色体鉴别药用植物丹参、甘西鼠尾的种子[J].安徽农业科学,2008,36(21)：9155-9156.
[10]Yan YP, Wang ZZ. Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method [J]. Plant Cell Tissue Organ Cult,2007,88:175-184.
[11]刘文婷,梁宗锁,蒋传中,等.丹参生殖生物学特性研究[J].现代中药研究与实践,2004,18(5)：17-20.
[12]Liu AH, Lin YH, Yang M, et al. High-performance liquid chromatographic determination of tanshinones in the roots of Salvia miltiorrhiza and related traditional Chinese medicinal preparations [J]. J. Pharm. Sci,2006,9(1):1-9.
[13]Wang X, Morris-Natschke SL, Lee KH. New developments in the chemistry and biology of the bioactive constituents of danshen [J]. Med. Res. Rev,2007,27 (1): 133-148.
[14]黄璐琦,戴住波,吕冬梅,等.探讨道地药材研究的模式生物及模型[J].中国中药杂志,2009,34(9)：1063-1066.
[15]王庆浩,陈爱华,张伯礼.丹参：一种中药研究的模式生物[J].中医药学报,2009,37(4)：1-4.
[16]Wang X, Geng Y, Li F, et al. Large-scale separation of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by pH-zone-refining countercurrent chromatography [J]. J. Sep. Sci,2007,30:3214-3217.
[17]Ma L J, Zhang XZ, Guo H, et al. Determination of four water-soluble compounds in Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometric electrode array system [J]. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci,2006,833:260-263.
[18]Lam FF, Yeung JH, Kwan YW, et al. Salvianolic acid B, an aqueous component of Danshen (Salvia miltiorrhiza), relaxes rat coronary artery by inhibition of calcium channels [J]. Eur. J. Pharmacol,2006,553:240-245.
[19]林汉钦.从丹参分离出一种新丹参酮成分[J].中华药学杂志,1993,45(9)：615-618.
[20]Choi J S, Kang H S, Jung H A, et al. A new cyclic phenyllactamide from Salvia miltiorrhiza [J]. Fitoterapia,2001,72(1):30-34.
[21]Zhou L, Chow M, Zuo Z. Improved quality control method for Danshen products-consideration of both hydrophilic and lipophilic active components [J]. J. Pharm. Biomed. Anal,2006,41(3):744-750.
[22]Liu YR, Qu SX, Maitz MF, et al. The effect of the major components of Salvia miltiorrhiza Bunge on bone marrow cells [J]. J. Ethnopharmacol,2007, 111(3):573-83.
[23]刘陶世,黄耀洲.丹参成分、制剂和质量控制研究概况[J].南京中医药大学学报,1998,14(4)：255-256.
[24]郭济贤.丹参的研究与临床应用[M].北京：中国医药科技出版社,1992：29.
[25]Li H C, Chang W L. Diterpenoids from Salvia miltiorrhiza [J]. Phytochemistry, 2000,53(8):951-953.
[26]Liu AH, Li L, Xu M, et al. Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by HPLC-DAD method [J]. J. Pharm. Biomed. Anal,2006,41:48-56.
[27]Zhang H, Yu C, Jia JY, et al. Contents of four active components in different commercial crude drugs and preparations of Danshen (Salvia miltiorrhiza) [J]. Acta. Pharmaco. Sin,2002,23:1163-1168.
[28]杜冠华,张均田.丹酚酸A对小鼠脑缺血再灌注致学习记忆功能障碍的改善作用及作用机制[J].药学学报,1995,30(3)：184.
[29]Li YG, Song L, Liu M, et al. Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma (Danshen) [J]. J. Chromatogr. A,2009,1216:1941-1953.
[30]Tsai MK, Lin YL, Huang YT. Effects of salvianolic acids on oxidative stress and hepatic fibrosis in rats [J]. Toxicol Appl Pharmacol,2010,242:155-164.
[31]Zhao GR, Zhang HM, Ye TX, et al. Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B[J]. Food Chem Toxicol, 2008,46:73-81.
[32]冯玲玲,周吉源.丹参的研究现状与应用前景[J].中国野生植物资源,2004,23(2)：4-7.
[33]陈震.丹参生长与隐丹参酮含量的关系[J].中药通报,1983,8(1)：2.
[34]文家富,王刚云,陈光华,等.商洛市丹参主要病虫害调查及综合防治技术[J].陕西农业科学,2009,1：210-211.
[35]Yuan JM, Tao LL, Xu JT. Immobilization of callus tissue cells of Salvia miltiorrhiza and the characteristics of their products [J]. Chin. J. Biotechnol,1990, 6(3):199-205.
[36]王康才,罗庆云,陈红霞,等.丹参愈伤组织中次生代谢产物形成的研究[J].中国中药杂志,1998,23(10)：592-594,638.
[37]Huang L, Liu D, Hu Z. Effects of phytohormones on growth and content of depsides in Salvia miltiorrhiza suspension cells [J]. Zhong Yao Cai (in Chinese), 2000,23(1):1-4.
[38]Wu JY, Ng J, Shi M, et al. Enhanced secondary metabolite (tanshinone) production of Salvia miltiorrhiza hairy roots in a novel root-bacteria coculture process. Appl Microbial Biotechnol,2007,77 (3):543-550.
[39]Shimomura K, Kitazawa T, Okamura N,et al. Tanshinone production in adventitious roots and regenerates of Salvia miltiorrhiza[J]. J. Nat. Prod,1991, 54(6):1583-1587.
[40]黄链栋,胡之壁,刘涤.丹参发状根再生植株的研究[J].上海中医药杂志,1996，10：40.
[41]张荫麟,宋经元,祁建军,等.农杆菌转化后丹参植株再生[J].中国中药杂志,1997,22(5)：274-276.
[42]宋经元,张荫麟,祁建军,等.丹参冠瘿组织丹参高产株系选择和丹参酮的产生[J].生物工程学报,1997,13(3)：317-319.
[43]Wang XY, Cui GH, Huang LQ, et al. Effects of methyl jasmonat on accumulation and release of tanshinones in suspension cultures of Salvia miltiorrhiza hairy root[J].中国中药杂志,2007,32(4)：300-302.
[44]Yan Q, Shi M, Ng J, et al. Elicitor-induced rosmarinic acid accumulation and secondary metabolism enzyme activities in Salvia miltiorrhiza hairy roots [J]. Plant Sci.2006,170:853-858.
[45]何水林,郑金贵,王晓峰,等.植物次生代谢：功能、调控及其基因工程[J].应用与环境生物学报,2002,8(5)：558-563.
[46]Yan YP, Wang ZZ, Tian W, et al. Generation and analysis of expressed sequence tags from the medicinal plant Salvia miltiorrhiza [J]. Sci. China Life Sci,2010,53: 273-285.
[47]崔光红,黄璐琦,邱德有,等.丹参功能基因组学研究Ⅱ—丹参毛状根不同时期基因表达谱分析[J].中国中药杂志,2007,32(13)：1267-1272.
[48]崔光红.丹参道地药材cDNA芯片构建及毛状根基因表达谱研究[D].北京：中国中医科学院,2006.
[49]Song J, Wang Z. Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmP4L1) from Salvia miltiorrhiza [J]. Mol. Biol. Rep,2009,36:939-52.
[50]刘世海.丹参肉桂酸4-羟化酶基因克隆及功能研究[D].陕西：陕西师范大学,2007.
[51]Huang B, Duan Y, Yi B, et al. Characterization and expression profiling of cinnamate 4-hydroxylase gene from Salvia miltiorrhiza in rosmarinic acid biosynthesis pathway[J]. Russ. J. Plant Physiol,2008,5:431-440.
[52]Zhao SJ, Hu ZB, Liu D, et al. Two divergent members of 4-coumarate:coenzyme a ligase from Salvia miltiorrhiza Bunge:cDNA cloning and functional study [J]. J. Integr. Plant Biol,2006,48:1355-1364.
[53]Huang B, Yi B, Duan Y, et al. Characterization and expression profiling of tyrosine aminotransferase gene from Salvia miltiorrhiza (Dan-shen) in rosmarinic acid biosynthesis pathway [J]. Mol. Biol. Rep,2008,35:601-612.
[54]段艳冰.丹参迷迭香酸代谢酪氨酸支路重要基因克隆及调控分析[D].上海：第二军医大学,2006.
[55]Xiao Y, Di P, Chen J, et al. Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (Smhppd) from Salvia miltiorrhiza hairy root cultures [J]. Mol. Biol. Rep,2009,36:2019-2029.
[56]Liao P, Zhou W, Zhang L, et al. Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Salvia miltiorrhiza [J]. Acta. Physiol. Plant,2009,31(3):565-572.
[57]化文平.丹参GGPP合酶基因的克隆及表达分析[D].陕西：陕西师范大学,2008.
[58]刘丛玲,王喆之.丹参晚期胚胎蛋白基因SmLEA的克隆及表达分析[J].生物技术通报,2009,5：80-84.
[59]刘世海,闫亚平,王喆之.丹参DHN2基因的克隆与序列分析[J].分子植物育种,2005,3(1)：66-70.
[60]王铭楷,闫亚平,田薇,等.丹参DHN1基因的克隆与序列分析[J].西北植物学报,2004,24(11)：1990-1995.
[61]张成,王喆之.丹参钙调蛋白cDNA的克隆及其反义表达载体的构建[J].分子植物育种,2006,4(3)：339-344.
[62]韩立敏.丹参APX和GPX基因克隆及其表达分析[D].陕西：陕西师范大学,2007.
[63]Tanaka T, Morimoto S, Nonaka G, et al. Magnesium and ammonium-potassium lithospermates B, the active principles having a uremia-preventive effect from Salvia miltiorrhiza[J]. Chem. Pharm. Bull,1989,37:340-344.
[64]Petersena M, Simmonds MS. Rosmarinic acid [J]. Phytochemistry,2003, 62:121-125.
[65]Petersen M, Ha'usler E, Karwatzki B, et al. Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth [J]. Planta,1993,189:10-14.
[66]Petersen M. Current status of metabolic phytochemistry[J]. Phytochemistry 2007; 68:2847-60.
[67]Grotewold E. Transcription factors for predictive plant metabolic engineering:are we there yet[J]. Curr. Opin. Biotechnol,2008,19:138-144.
[68]Stracke R, Werber M, Weisshaar B. The R2R3-MYB gene family in Arabidopsis thaliana [J]. Curr. Opin. Plant. Biol,2001,4:447-456.
[69]Wilkins O, Nahal H, Foong J, et al. Expansion and diversification of the populus R2R3-MYB family of transcription factors [J]. Plant Physiol,2009,149:981-993.
[70]Hemm MR, Herrmann KM, Chapple C. AtMYB4:a transcription factor general in the battle against UV [J]. Trends Plant Sci,2001,6:135-136.
[71]Preston J, Wheeler J, Heazlewood J, et al. AtMYB32 is required for normal pollen development in Arabidopsis thaliana [J]. Plant J,2004,40:979-995.
[72]Borevitz J O, Xia Y J, Blount J, et al. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis [J]. Plant Cell,2000,12: 2383-2393.
[73]Galis I, Simek P, Narisawa T, et al. A novel R2R3 MYB transcription factor NtMYBJSl is a methyl jasmonate-dependent regulator of phenylpropanoid conjugate biosynthesis in tobacco [J]. Plant J,2006,46:573-592.
[74]Ritter H, Schulz GE. Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase [J]. Plant Cell,2004,16: 3426-3436.
[75]Lois R, Dietrich A, Hahlbrock K, et al. A phenylalanine ammonia-lyase gene from parsley:structure, regulation and identification of elicitor and light responsive cis-acting elements [J]. EMBO J,1989,8:1641-1648.
[76]Dixon RA, Paiva NL. Stress-induced phenylpropanoid metabolism [J]. Plant Cell, 1995,7:1085-1097.
[77]MacDonald MJ, D'Cunha GB. A modern view of phenylalanine ammonia lyase [J]. Biochem Cell Biol,2007,85:273-282.
[78]Shadle GL, Wesley SV, Korth KL, et al. Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase [J]. Phytochemistry,2003,64:153-161.
[79]Ohl S, Hedrick SA, Chory J, et al. Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis [J]. Plant Cell,1990,2:837-848.
[80]Yeo YS, Lee SW, Kim YH, et al. Restriction mapping of phenylalanine ammonia-lyase gene family in tomato(Lycopersicon esculentum) [J]. RDA J. Agric. Sci. Biotechnol,1994,36(2):187-192.
[81]Pellegrini L, Rohfritsch O, Fritig B, et al. Phenylalanine ammonia-lyase in tobacco: molecular cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the response to a fungal elicitor [J]. Plant Physiol,1994, 106:877-886.
[82]Elkind Y, Edwards R, Mavandad M, et al. Abnormal plant development and down-regulation of phenylpropanoid biosynthesis in transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene [J]. Proc. Nadl. Acad. Sci,1990, 81:9057-9061.
[83]Chen F, Reddy MS, Temple S, et al. Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wallbound ferulic acid in alfalfa (Medicago sativa L.) [J]. Plant J,2006,48:113-124.
[84]Rohde A, Morreel K, Ralph J, et al. Molecular phenotyping of the pall and pal2 mutants of Arabidopsis Thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism [J]. Plant Cell,2004, 16:2749-2771.
[85]Mahesh V, Rakotomalala JJ, Gal LL, et al. Isolation and genetic mapping of a Coffea canephora phenylalanine ammonia-lyase gene (CcPAL1) and its involvement in the accumulation of caffeoyl quinic acids [J]. Plant Cell Rep,2006, 25(9):986-992.
[86]Liu R, Xu S, Li J, et al. Expression profile of a PAL gene from Astragalus membranaceus var. mongholicus and its crucial role in flux into flavonoid biosynthesis [J]. Plant Cell Rep,2006,25(7):705-710.
[87]Razzaque A, Ellis BE. Rosmarinic acid production in Coleus cell cultures [J]. Planta,1977,137:287-291.
[88]Chen H, Chen F. Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures [J]. Plant Cell Rep,2000, 19:710-717.
[89]Morant M, Bak S, Moller BL, et al. Plant cytochromes P450:tools for pharmacology, plant protection and phytoremediation [J]. Curr Opin Biotechnol, 2003,14(2):151-162.
[90]Chen AH, Chai YR, Li JN, et al. Molecular cloning of two genes encoding cinnamate 4-hydroxylase (c4h) from oilseed rape (Brassica napus) [J]. J. Biochem. Mol. Biol,2007,40:247-260.
[91]Blount JW, Korth KL, Masoud SA, et al. Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway [J]. Plant Physiol,2000,122: 107-116.
[92]Ro DK, Mah N, Ellis BE, et al. Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase [J]. Plant Physiol,2001,126(1):317-329.
[93]Koopmann E, Logemann E, Hahlbrock K. Regulation and functional expression of cinnamate 4-hydroxylase from parsley [J]. Plant Physiol,1999,119(1):49-56.
[94]Teutsch HG, Hasenfratz MP, Lesot A, et al. Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway [J]. Proc. Natl. Acad. Sci. USA,1993,90(9):4102-4106.
[95]Ranjeva R, Boudet A, Faggiox R. Phenolic metabolism in petunia tissues. IV. Properties of p-coumarate:coenzyme a ligase isoenzymes [J]. Biochimie,1976,58: 1255-1262.
[96]Ehlting J, Buttner D, Wang Q, et al. Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms [J]. Plant J,1999,19:9-20.
[97]Hu WJ, Kawaoka A, Tsai CJ, et al. Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides) [J]. Proc. Natl. Acad. Sci. USA,1998,95,5407-5412.
[98]Kumer A, Ellis BE.4-Coumarate:CoA ligase gene family in Rubus idaeus:cDNA structures, evolution, and expression [J]. Plant Mol. Biol,2003,31:327-340.
[99]Hamberger B, Hahlbrock K. The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapateactivating and three commonly occurring isoenzymes [J]. Proc. Nadl. Acad. Sci,2004,101:2209-2214.
[100]De Eknamkul W, Ellis BE. Tyrosine aminotransferase:the entrypoint enzyme of the tyrosine-derived pathway in rosmarinic acid biosynthesis [J]. Phytochemistry, 1987,26:1941-1946.
[101]Heike HC, Janine G, Iris S, et al. Tocopherol content and activities of tyrosine aminotransferase and cystine lyase in Arabidopsis under stress conditions [J]. J. Plant Physiol,2005,162:767-770.
[102]Petersen M, Alfermann AW. Two new enzymes of rosmarinic acid biosynthesis from cell cultures of Coleus blumei:hydroxyphenylpyruvate reductase and rosmarinic acid synthase [J]. Z Naturforsch,1988,43c:501-504.
[103]Kim KH, Janiak V, Petersen M. Purification,cloning and functional expression of hydroxyphenylpyruvate reductase involved in rosmarinic acid biosynthesis in cell cultures of Coleus blumei [J]. Plant Mol Biol,2004,54:311-323.
[104]肖莹.丹参酚酸类成分生源合成的调控研究[D].上海：第二军医大学,2009.
[105]Berger A, Meinhard J, Petersen M. Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases [J]. Planta,2006,224:1503-1510.
[106]St. Pierre B, De Luca V. Evolution of acyltransferase genes:origin and diversification of the BAHD superfamily of acyltransferases involved in secondary metabolism [J]. Rec. Adv. Phytochem,2000,34:285-316.
[107]D'Auria JC. Acyltransferases in plants:a good time to be BAHD [J]. Curr. Opin. Plant. Biol,2006,9:331-340.
[108]St. Pierre B, LaXamme P, Alarco AM, et al. The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A dependent acyl transfer [J]. Plant J,1998,14:703-713.
[109]Dudareva N, D'Auria JC, Nam KH, et al. Acetyl-CoA:benzylalcohol acetyltransferase — an enzyme involved in floral scent production in Clarkia breweri [J]. Plant J,1998,14:297-304.
[110]Hoffmann L, Maury S, Martz F, et al. Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism [J]. J. Biol. Chem,2003,278:95-103.
[111]Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcine synthase gene into Petunia results in reversible co-suppression of homologous genes in trans [J]. Plant Cell,1990,2:279-289.
[112]Fire A, Xu SQ, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature,1998, 39(1):806-811.
[113]何承伟,刘芳,刘新光,等.RNA干涉作用机制的研究进展[J].医学综述,2005,11(4)：291-294.
[114]徐秉良,师桂英,王延秀.RNA干扰与基因敲除[J].生物技术通报,2004,15(4)：386-388.
[115]孙建国,廖荣霞,陈正堂.RNA干涉分子机制研究进展[J].生物化学与生物 物理进展,2002,29(5)：678-680.
[116]周小云,陈信波,向建华.RNA技术及在植物功能基因组研究中的应用[J].生物学杂志,2005,22(2)：38-41.
[117]Bezanilla M, Pan A, Ralph SQ. RNA interference in the moss physcomitrella patens [J]. Plant physiol,2003,133(2):470-474.
[118]Shinjiro O, Hirotaka U, Yube Y, et al. Producing decaffeinated coffee plants [J]. Nature,2003,423(19):823.
[119]Gianfranco D, Raffaela T, Ralf W, et al. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase [J]. BMC Plant Biol,2006,6:13.
[120]Xiong AS, Yao QH, Peng RH, et al. Diferent effection ACC oxidase gene silencing triggered by RNA interference in transgenic tomato [J]. Plant Cell Rep, 2005,23:639-646.
[121]Coleman HD, Park J, Nair R, et al. RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism [J]. Proc. Nadl. Acad. Sci,2008,105:4501-4506.
[122]栾雨时,包永明.生物工程实验技术手册[M].北京：化学工业出版社,2005：167-168.
[123]Baxevanis A D, Ouellette B F F(著),李衍达,孙之荣等(译).生物信息学[M].北京：清华大学出版社,2000：231-250.
[124]Mount D W(著),钟扬,王莉,等.生物信息学[M].北京：高等教育出版社,2003：301-345.
[125]Kyte J, Doolittler F. A simple method for displaying the hydropathic character of a protein [J]. J. Mol. Biol,1982,157 (6):105-132.
[126]Nielsen H, Engelbrecht J, Brunak S, et al. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites [J]. Protein Eng, 1997,10 (1):1-6.
[127]Bendtsen J D, Nielsen H, Heijne G V, et al. Prediction of signal peptides:Signal P 3.0 [J]. J. Mol. Biol,2004,340:783-795.
[128]翟中和,王喜忠,丁明孝.细胞生物学[M].北京：高等教育出版社,2000：323-328.
[129]王镜岩,朱圣庚,徐长法(主编).生物化学(上册)(第三版)[M].北京：高等教育出版社,2002：157-181.
[130]Geourjon C, Deleage G. SOPMA:Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments [J]. Comput. Appl. Biosci,1995,11 (6):681-684.
[131]Nakazawa A, Nozue M, Yasuda H, et al. Expression pattern and gene structure of phenylalanine ammonia-lyase in Pharbitis nil [J]. J. Plant Res,2001,114:323-328.
[132]Logemann E, Parniske M, Hahlbrock K. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley [J]. Proc. Natl. Acad. Sci. USA,1995,92:5905-5909.
[133]Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture [J]. Physiol Plant,1962,15:473-497.
[134]闫亚平.基于EST技术的丹参大规模基因克隆及其外源基因转化技术研究[D].陕西：陕西师范大学,2005.
[135]Wesley SV, Helliwell CA, Smith NA, et al. Construct design for efficient, effective and high throughput gene silencing in plants [J]. Plant J,2001,27: 581-590.
[136]Gleave AP. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome [J]. Plant Mol Biol,1992,20:1203-1207.
[137]Lee HS, Wicker L. Anthocyanin pigments in the skin of lychee fruit [J]. J. Food Sci,1991,56(2):466-468,483.
[138]Dewanto V, Wu X, Adorn KK, et al. Thermal proeessing enhances the nutritional value of tomatoes by inereasing total antioxidant activity [J]. J. Agric. Food Chem, 2002,50:3010-3014.
[139]Brand-Williams W, Cuvelier ME, Berset C. Use of free radical method to evaluate antioxidant activity [J]. Lebensm. Wiss. Technol,1995,28:25-30.
[140]Dorman H, Peltoketoa A, Hiltunena R, et al. Characterisation of the antioxidant properties of de-odourised aqueous extracts from selected Lamiaceae herbs [J]. Food Chemistry,2003,83:255-262.
[141]Jin H, Cominelli E, Bailey P, et al. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis [J]. EMBO J,2000, 19:6150-6161.
[142]Petersen M, Abdullah Y, Benner J, et al. Evolution of rosmarinic acid biosynthesis [J]. Phytochemistry,2009,70:1663-1679.
[143]Nakayama T, Suzuki H, Nishino T. Anthocyanin acyltransferases:specificities, mechanism, phylogenetics, and applications [J]. J. Mol. Catal., B Enzym,2003,23: 117-132.
[144]Tacke E, Korfhage C, Michel D, et al. Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm [J]. Plant J,1995,8:907-917.
[145]Negruk V, Yang P, Subramanian M, et al. Molecular cloning and characterization of the CER2 gene of Arabidopsis thaliana [J]. Plant J,1996,9:137-145.
[146]Xia Y, Nikolau BJ, Schnable PS. Cloning and characterization of CER2, an Arabidopsis gene that affects cuticular wax accumulation [J]. Plant Cell,1996,8: 1291-1304.
[147]Bayer A, Ma XY, Stockigt J. Acetyltransfer in natural product biosynthesis— functional cloning and molecular analysis of vinorine synthase [J]. Bioorg Med. Chem,2004,12:2787-2795.
[148]Shalit M, Guterman I, Volpin H, et al. Volatile ester formation in roses. Identification of an acetyl-coenzyme A geraniol/citronellol acetyltransferase in developing rose petals [J]. Plant Physiol,2003,131:1868-1876.
[149]Boatright J, Negre F, Chen XL, et al. Understanding in vivo benzenoid metabolism in Petunia petal tissue [J]. Plant Physiol,2004,135:1993-2011.
[150]Wang J, De Luca V. The biosynthesis and regulation of biosynthesis of Concord grape fruit esters, including'foxy'methyl anthranilate [J]. Plant J,2005, 44:606-619.
[151]Wagner A, Ralph J, Akiyama T, et al. Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiate [J]. Proc. Natl. Acad. Sci. USA,2007,104:11856-11861
[152]Comino C, Hehn A, Moglia A, et al. The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway [J]. BMC Plant Biol,2009,9:30.
[153]Rischer H, Oresic M, Seppanen-Laakso T, et al. Gene-tometabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells [J]. Proc. Natl. Acad. Sci. USA,2006,103:5614-5619.
[154]Zulak KG, Cornish A, Daskalchuk TE, et al. Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism [J]. Planta,2007,225:1085-1106.
[155]Xiao Y, Gao S, Di P, et al. Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures [J]. Physiol. Plant,2009,137:1-9.