丹参酮代谢途径DXR基因的克隆、特性分析及功能初步验证和DXSI基因启动子的克隆
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摘要
丹参(Salvia miltiorrhira),是唇形科鼠尾草属的多年生草本植物。作为一种传统的中药材,丹参在临床上常被用来治疗心绞痛,心肌梗塞等心血管疾病。丹参的活性成分可分为水溶性和脂溶性活性成分。大多数脂溶性的活性成分是丹参酮形式的二萜醌类化合物,包括丹参酮Ⅰ,丹参酮ⅡA,丹参酮ⅡB,隐丹参酮以及其他相关成分。
萜类是植物界中广泛存在的一类次生代谢物质,也是目前最大的一类植物天然产物。所有天然萜类化合物(包括丹参酮)均来自于两个通用5碳前体异戊烯基焦磷酸(IPP)和二甲基烯丙基焦磷酸(DMAPP)。其生物合成途径有两条：甲羟戊酸(mevalonate, MVA)和1-去氧木糖-5-磷酸(1-deoxy-Dxylulose-5-phosphate,DXP)途径。
本研究以丹参为材料,采用RACE法克隆丹参酮代谢途径(包含DXP途径)中5-磷酸脱氧木酮糖还原异构酶(DXR)基因(DXR, GenBank Accession No.FJ476255)。SmDXR全长cDNA包含1425bp的开放阅读框,编码475个氨基酸。氨基酸多重序列比对结果显示SmDXR与其他植物的DXR如薄荷(Mentha x piperita)金鱼草(Antirrhinum majus)等有比较高的相似性,分别达到98%和91%。进化树表明,SmDXR与番茄(Lycopersicon esculentum)的DXR有着最近的亲缘关系。组织表达分析表明SmDXR都能在根茎叶中表达：在叶中表达最高,其次是根和茎,暗示SmDXR是一个组成型表达的基因,这和大多数报道的DXR基因一致。水杨酸(SA)诱导实验表明：SmDXR能够被SA诱导。甲基茉莉酸(MeJA)诱导实验表明,SmDXR不被MeJA诱导。通过大肠杆菌(E.coli)颜色互补实验表明：SmDXR编码了一个有功能的蛋白,同时SmDXR能够推动DXP代谢途径的流动。此外,本研究还通过LA-PCR的方法克隆得到丹参酮代谢途径中5-磷酸脱氧木酮糖合成酶Ⅰ(DXSI)基因的5'UTR及启动子区域862bp片段。通过PlantCARE网站预测,该序列除了含有启动子的基本元件TATA-box和CAAT-box外,还含有一些胁迫诱导元件,如ABRE：与脱落酸响应的顺式作用元件；CGTCA-motif:与甲基茉莉酸响应的顺式作用元件；GARE-motif:与赤霉素响应的顺式作用元件；TCA-element:与水杨酸响应的顺式作用元件；TGA-box:与生长素响应的顺式作用元件等。
Salvia miltiorrhira, is a kind of species which belongs to family Labiatae, genera Salvia. As a kind of important ancient Chinese medicine, Salvia miltiorrhira is used to treat cardiovascular diseases such as particularly angina pectoris and myocardial infarction. On the basis of the chemical structures, the major bioactive components in S. miltiorrhiza BUNGE can be classified as lipid-soluble diterpenoids and water-soluble polyphenolic compounds. Most of lipophilic constituents are diterpene chinone compounds of the tanshinone type, including tanshinoneⅠ, tanshinoneⅡA, tanshinone IIB, cryptotanshinone and so on.
Terpenes are one kind secondary metabolites which widespread in plants while they are also the largest groups of natural products comprising numerous compounds. All the terpenes are formed through the reactions of IPP (isopentenyl pyrophosphate) and DMAPP (dimethylallyl pyrophosphate), which via two biological reactions, MVA pathway and DXP pathway.
The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255) by the method of RACE. The cloned full-length cDNA of contained a 1425bp ORF encoding a deduced protein of 475 amino acid residues. Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. On the amino acid level, SmDXR was 98% and 91% identical to Mentha x piperita DXR and Antirrhinum majus DXR. According to the phylogenetic tree, SmDXR belongs to the plant group and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression analysis showed that SmDXR was differentially expressed in various tissues and was found to be higher in leaves followed by roots and stems, indicating SmDXR was considered to be a constitutively expressing gene, which is similar with the most DXRs which have been reported. Expression analysis of SmDXR under induction of SA and MeJA showed that SmDXR can be induced by SA while can not be induced by MeJA. Color complementation assay confirmed SmDXR encoded a functional protein and played an important role in promoting DXP pathway flux.
Additionally, the DXSI's five prime untranslated region and promoter sequence of 862bp was cloned for the first time by LA-PCR. The predicting result of PlantCARE website indicates that the sequence has not only some basic elements including TATA-box and CAAT-box but also some stress-induced elements, for example, ABRE (cis-acting element involved in the abscisic acid responsiveness), cis-acting regulatory element involved in the MeJA-responsiveness, CGTCA-motif (cis-acting regulatory element involved in the MeJA-responsiveness), GARE-motif (gibberellin-responsive element), TCA-element (cis-acting element involved in salicylic acid responsiveness) and so on.
萜类是植物界中广泛存在的一类次生代谢物质,也是目前最大的一类植物天然产物。所有天然萜类化合物(包括丹参酮)均来自于两个通用5碳前体异戊烯基焦磷酸(IPP)和二甲基烯丙基焦磷酸(DMAPP)。其生物合成途径有两条：甲羟戊酸(mevalonate, MVA)和1-去氧木糖-5-磷酸(1-deoxy-Dxylulose-5-phosphate,DXP)途径。
本研究以丹参为材料,采用RACE法克隆丹参酮代谢途径(包含DXP途径)中5-磷酸脱氧木酮糖还原异构酶(DXR)基因(DXR, GenBank Accession No.FJ476255)。SmDXR全长cDNA包含1425bp的开放阅读框,编码475个氨基酸。氨基酸多重序列比对结果显示SmDXR与其他植物的DXR如薄荷(Mentha x piperita)金鱼草(Antirrhinum majus)等有比较高的相似性,分别达到98%和91%。进化树表明,SmDXR与番茄(Lycopersicon esculentum)的DXR有着最近的亲缘关系。组织表达分析表明SmDXR都能在根茎叶中表达：在叶中表达最高,其次是根和茎,暗示SmDXR是一个组成型表达的基因,这和大多数报道的DXR基因一致。水杨酸(SA)诱导实验表明：SmDXR能够被SA诱导。甲基茉莉酸(MeJA)诱导实验表明,SmDXR不被MeJA诱导。通过大肠杆菌(E.coli)颜色互补实验表明：SmDXR编码了一个有功能的蛋白,同时SmDXR能够推动DXP代谢途径的流动。此外,本研究还通过LA-PCR的方法克隆得到丹参酮代谢途径中5-磷酸脱氧木酮糖合成酶Ⅰ(DXSI)基因的5'UTR及启动子区域862bp片段。通过PlantCARE网站预测,该序列除了含有启动子的基本元件TATA-box和CAAT-box外,还含有一些胁迫诱导元件,如ABRE：与脱落酸响应的顺式作用元件；CGTCA-motif:与甲基茉莉酸响应的顺式作用元件；GARE-motif:与赤霉素响应的顺式作用元件；TCA-element:与水杨酸响应的顺式作用元件；TGA-box:与生长素响应的顺式作用元件等。
Salvia miltiorrhira, is a kind of species which belongs to family Labiatae, genera Salvia. As a kind of important ancient Chinese medicine, Salvia miltiorrhira is used to treat cardiovascular diseases such as particularly angina pectoris and myocardial infarction. On the basis of the chemical structures, the major bioactive components in S. miltiorrhiza BUNGE can be classified as lipid-soluble diterpenoids and water-soluble polyphenolic compounds. Most of lipophilic constituents are diterpene chinone compounds of the tanshinone type, including tanshinoneⅠ, tanshinoneⅡA, tanshinone IIB, cryptotanshinone and so on.
Terpenes are one kind secondary metabolites which widespread in plants while they are also the largest groups of natural products comprising numerous compounds. All the terpenes are formed through the reactions of IPP (isopentenyl pyrophosphate) and DMAPP (dimethylallyl pyrophosphate), which via two biological reactions, MVA pathway and DXP pathway.
The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255) by the method of RACE. The cloned full-length cDNA of contained a 1425bp ORF encoding a deduced protein of 475 amino acid residues. Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. On the amino acid level, SmDXR was 98% and 91% identical to Mentha x piperita DXR and Antirrhinum majus DXR. According to the phylogenetic tree, SmDXR belongs to the plant group and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression analysis showed that SmDXR was differentially expressed in various tissues and was found to be higher in leaves followed by roots and stems, indicating SmDXR was considered to be a constitutively expressing gene, which is similar with the most DXRs which have been reported. Expression analysis of SmDXR under induction of SA and MeJA showed that SmDXR can be induced by SA while can not be induced by MeJA. Color complementation assay confirmed SmDXR encoded a functional protein and played an important role in promoting DXP pathway flux.
Additionally, the DXSI's five prime untranslated region and promoter sequence of 862bp was cloned for the first time by LA-PCR. The predicting result of PlantCARE website indicates that the sequence has not only some basic elements including TATA-box and CAAT-box but also some stress-induced elements, for example, ABRE (cis-acting element involved in the abscisic acid responsiveness), cis-acting regulatory element involved in the MeJA-responsiveness, CGTCA-motif (cis-acting regulatory element involved in the MeJA-responsiveness), GARE-motif (gibberellin-responsive element), TCA-element (cis-acting element involved in salicylic acid responsiveness) and so on.
引文
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[2]唐克轩.中草药生物技术.2005：331
[3]韩军丽,李振秋,刘本叶,王红,李国凤,叶和春.植物萜类代谢工程.植物工程学报.2007,23(4)：561-569
[4]肖培根.中药植物原色图鉴.1999：413
[5]Zhou L, Zuo Z, Chow MS. Danshen:an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharma-col.2005,45:1345-1359
[6]郭增军.丹参提取物中有效成分的质量控制及药动学研究 2008年沈阳药科大学博士论文
[7]Lee DS, Lee SH, Noh JG, Hong SD. Antibacterial activities of cryptotanshinone and dihydrotanshinone I from a medicinal herb, Salvia miltiorrhiza Bunge. Biosci Biotechnol BioChem.1999,63(12):2236-9.
[8]Feng H, Xiang H, Zhang J, Liu G, Guo N, Wang X, Wu X, Deng X, Yu L. Genome-Wide Transcriptional Profiling of the Response of Staphylococcus aureus to Cryptotanshinone. J Biomed Biotechnol.2009:617509
[9]Niu XL, Ichimori K, Yang X, Hirota Y, Hoshiai K, Li M, Nakazawa H. Tanshinone II-A inhibits low density lipoprotein oxidation in vitro. Free Radic Res.2000,33(3):305-312.
[10]Takahashi K, Ouyang X, Komatsu K, Nakamura N, Hattori M, Baba A, Azuma Sodium tanshinone ⅡA sulfonate derived from Danshen (Salvia miltiorrhiza) attenuates hypertrophy induced by angiotensin Ⅱ in cultured neonatal rat cardiac cells. J Bichem Pharmacol.2002, 64(4):745-749.
[11]Lu Q, Zhang P, Zhang X, Chen J. Experimental study of the anti-cancer mechanism of tanshinone IIA against human breast cancer. Int J Mol Med.2009,24(6):773-780.
[12]Shan YF, Shen X, Xie YK, Chen JC, Shi HQ, Yu ZP, Song QT, Zhou MT, Zhang QY. Inhibitory effects of tanshinone Ⅱ-A on invasion and metastasis of human colon carcinoma cells. Acta Pharmacol Sin.2009,30(11):1537-42.
[13]Dong X, Dong J, Zhang R, Fan L, Liu L, Wu G. Anti-inflammatory effects of tanshinone IIA on radiation-induced microglia BV-2 cells inflammatory response. Cancer Biother Radiopharm.2009,24(6):681-7.
[14]Jin DZ, Yin LL, Ji XQ, Zhu XZ. Cryptotanshinone inhibits cyclooxygenase-2 enzyme activity but not its expression. Eur J Pharmacol.2006,549:166-172
[15]Park EJ, Zhao YZ, Kim YC, Sohn DH. Preventive effects of a purified extract isolated from Salvia miltiorrhiza enriched with tanshinone Ⅰ, tanshinone ⅡA and cryptotanshinone on hepatocyte injury in vitro and in vivo. Food Chem Toxicol.2009,47(11):2742-8.
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