雷公藤4个环氧角鲨烯环化酶基因克隆与表达分析
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摘要
雷公藤(Tripterygium wilfordii)作为重要的药用植物,具有抗癌、抗炎和免疫调节等作用,并具有较强的杀虫活性。雷公藤红素(celastrol)是雷公藤最具代表性的三萜物质,药理活性高且作用广泛,极具开发价值。环氧角鲨烯环化酶(oxidosqualene cyclase, OSC)被认为是三萜类次生代谢途径中的关键限速酶,是三萜合成生物学研究的重要元件之一。根据前期对雷公藤根转录组数据的分析设计引物,克隆得到4个雷公藤OSC基因:TwOSC1 (GenBank No. MH412928)、TwOSC2 (GenBank No. MH412929)、TwOSC3(GenBank No. MH412930)和TwOSC4 (GenBank No. MH412931)。TwOSC1～3的ORF均为2 289 bp,编码762个氨基酸;TwOSC4的ORF为2 280 bp,编码759个氨基酸。生物信息学分析表明,4个TwOSC蛋白序列相似性较高,多重序列两两比对相似性在53%～77%之间。分子进化树分析表明,TwOSC1与多个物种的β-香树素合成酶聚为一个分支,TwOSC2、TwOSC4与环阿屯醇合成酶聚为一个分支,TwOSC3与羽扇豆醇合成酶聚为一个分支。以qRT-PCR方法检测上述基因在雷公藤不同组织部位的表达模式,结果显示,TwOSC1基因只在根中表达,TwOSC2、TwOSC3和TwOSC4在不同组织中均有表达。进而以茉莉酸甲酯(methyl jasmonate, MeJA)诱导处理雷公藤发状根,可不同程度地增加TwOSC1、TwOSC2、TwOSC4基因表达,同时抑制TwOSC3基因表达。以高效液相色谱法(high-performance liquid chromatography,HPLC)检测雷公藤发状根中红素的含量变化,经MeJA诱导处理后红素含量有不同程度增加,且MeJA诱导后TwOSC1基因表达量的变化趋势与红素含量的变化趋势一致,推测TwOSC1可能与雷公藤红素的合成有关。本研究为雷公藤三萜活性物质的合成与积累提供了基础资料。
Tripterygium wilfordii is an important medicinal plant that has anti-cancer, anti-inflammatory and immune system regulation effect, and also has strong insecticidal activity. Celastrol is the most representative triterpenoid in T. wilfordii which has high pharmacological activity for medical application and potential development value. Oxidosqualene cyclase(OSC) is considered to be the key rate-limiting enzyme in the triterpenoid biosynthesis pathway. The present study cloned 4 OSC genes, TwOSC1(GenBank No.MH412928), TwOSC2(GenBank No. MH412929), TwOSC3(GenBank No. MH412930) and TwOSC4(GenBank No. MH412931), from T. wilfordii. The ORFs of TwOSC1, TwOSC2 and TwOSC3 were all 2 289 bp and encoded 762 amino acids. The TwOSC4 ORF was 2 280 bp and encoded 759 amino acids. Bioinformatics analysis showed that the amino acid sequence similarity of the 4 TwOSCs was high, and the similarity of multiple sequence alignments between each 2 sequences was 53% to 77%. Phylogenetic analysis showed that TwOSC1 clustered with β-amyrin synthase, TwOSC2 and TwOSC4 clustered with cycloartenol synthase, and TwOSC3 clustered with lupeol synthase. The expression patterns of 4 TwOSC genes in different tissues and the expression level in T. wilfordii hairy roots treated by methyl jasmonate(MeJA) were detected by qRT-PCR.TwOSC1 had a very strong tissue-specificity and was only expressed in the root. However, TwOSC2, TwOSC3,and TwOSC4 were expressed in all tissues. The expression level of TwOSC1, TwOSC2 and TwOSC4 increased by different degrees in T. wilfordii hairy roots after MeJA treatment. But MeJA treatment inhibited the expression of TwOSC3. The content of celastrol that was detected by high-performance liquid chromatography(HPLC) increased after MeJA treatment in T. wilfordii hairy roots. The change of TwOSC1 expression was consistent with the change of celastrol content after MeJA treatment. Above results indicate that TwOSC1 may be involved in the synthesis of celastrol in T. wilfordii hairy roots. The present study provides some basic data for the synthesis and regulation of triterpenoid in T. wilfordii.
Tripterygium wilfordii is an important medicinal plant that has anti-cancer, anti-inflammatory and immune system regulation effect, and also has strong insecticidal activity. Celastrol is the most representative triterpenoid in T. wilfordii which has high pharmacological activity for medical application and potential development value. Oxidosqualene cyclase(OSC) is considered to be the key rate-limiting enzyme in the triterpenoid biosynthesis pathway. The present study cloned 4 OSC genes, TwOSC1(GenBank No.MH412928), TwOSC2(GenBank No. MH412929), TwOSC3(GenBank No. MH412930) and TwOSC4(GenBank No. MH412931), from T. wilfordii. The ORFs of TwOSC1, TwOSC2 and TwOSC3 were all 2 289 bp and encoded 762 amino acids. The TwOSC4 ORF was 2 280 bp and encoded 759 amino acids. Bioinformatics analysis showed that the amino acid sequence similarity of the 4 TwOSCs was high, and the similarity of multiple sequence alignments between each 2 sequences was 53% to 77%. Phylogenetic analysis showed that TwOSC1 clustered with β-amyrin synthase, TwOSC2 and TwOSC4 clustered with cycloartenol synthase, and TwOSC3 clustered with lupeol synthase. The expression patterns of 4 TwOSC genes in different tissues and the expression level in T. wilfordii hairy roots treated by methyl jasmonate(MeJA) were detected by qRT-PCR.TwOSC1 had a very strong tissue-specificity and was only expressed in the root. However, TwOSC2, TwOSC3,and TwOSC4 were expressed in all tissues. The expression level of TwOSC1, TwOSC2 and TwOSC4 increased by different degrees in T. wilfordii hairy roots after MeJA treatment. But MeJA treatment inhibited the expression of TwOSC3. The content of celastrol that was detected by high-performance liquid chromatography(HPLC) increased after MeJA treatment in T. wilfordii hairy roots. The change of TwOSC1 expression was consistent with the change of celastrol content after MeJA treatment. Above results indicate that TwOSC1 may be involved in the synthesis of celastrol in T. wilfordii hairy roots. The present study provides some basic data for the synthesis and regulation of triterpenoid in T. wilfordii.
引文
陈铭祥,冯玉静,王定勇,等.2010.雷公藤红素的研究进展[J].中成药,32(3):473-476.(Chen M X,Feng Y JWang D Y,et al.2010.Research progress of celastro[J].Chinese Traditional Patent Medicine,32(3):473-476.)
罗都强,张兴,冯俊涛.2000.杀虫植物雷公藤研究进展[J]西北农林科技大学学报(自然科学版),28(3):84-89(Luo D Q,Zhang X,Feng J T.2000.Research development of an insecticidal plant Tripterygium wilfordii Hook[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry(Natural Science Edition),28(3)84-89.)
梁新华,樊平.2016.雷公藤多苷片对糖尿病肾脏病变的临床疗效系统评价[J].山西医科大学学报,47(8):751-755.(Liang X H,Fan P.2016.Systemic evaluation of clinical effects of Tripterygium glycosides on diabetic nephropathy[J].Journal of Shanxi Medical University47(8):751-755.)
刘佳,付爽,王萌,等.2017.低温胁迫下人参愈伤组织中氧化鲨烯环化酶基因家族的表达特性分析[J].北方园艺,41(17):164-168.(Liu J,Shuang F U,Wang M,et al2017.Effect of chilling stress on expression characteristic of oxidosqualene cyclase(OSC)in callus of Panax ginseng[J].Northern Horticulture,41(17):164-168.)
任献青,鲁静,孟祥乐,等.2013.雷公藤红素药理作用最新研究进展[J].中华中医药杂志,28(9):2679-2682.(Ren X Q,Lu J,Meng X L,et al.2013.Recent progress on pharmacological effects of celastrol[J].China Journal of Traditional Chinese Medicine and Pharmacy,28(9):2679-2682.)
沈湛云,刘春生,王学勇.2009.甘草β-香树酯醇合成酶的编码区克隆与序列分析[J].中国中药杂志,2009,34(19):2438-2440.(Shen Z Y,Liu C S,Wang X Y.2009.Cloning and characterization of open reading frame encodingβ-amyrin synthase in Glycyrrhiza uralensis[J].China Journal of Chinese Materia Medica,34(19):2438-2440.)
夏焱,段宏泉,张铁军,等.2005.雷公藤属药用植物的研究进展[J].中草药,36(7):1093-1096.(Xia Y,Duan H Q,Zhang T J,et al.2005.Advances in studies on medicinal plants of Tripterygium Hook.f.[J].Chinese Traditional and Herbal Drugs,36(7):1093-1096.)
袁凯.2017.雷公藤甲素、雷公藤红素通过调节中性粒细胞活性治疗类风湿关节炎的机制研究[D].博士学位论文,北京中医药大学,导师:徐安龙,pp.18-23.(Yuan K.2017.Triptolide and celastrol treatment of rheumatoid arthritis by regulating neutrophil activity[D].Thesis for Ph.D.,Beijing University of Chinese Medicine,Supervisor:Xu A L,pp.18-23.)
Basyuni M,Oku H,Tsujimoto E,et al.2010.Triterpene synthases from the Okinawan mangrove tribe Rhizophoraceae[J].FEBS Journal,274(19):5028-5042.
Brinker A M,Ma J,Lipsky P E,et al.2007.Medicinal chemistry and pharmacology of genus Tripterygium(Celastraceae)[J].Phytochemistry,38(27):732-766.
Brinker A M,Raskin I.2005.Determination of triptolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reverse phase high-performance liquid chromatography[J].Journal of Chromatography A,1070(1-2):65-70.
Choi D W,Jung J D,Ha Y I,et al.2005.Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites[J].Plant Cell Reports,23(8):557-566.
Chang C H,Chen Y C,Tseng S W,et al.2012.The cysteine703 to isoleucine or histidine mutation of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae generates an iridal-type triterpenoid[J].Biochimie,94(11):2376-2381.
García I,Castellano J M,Vioque B,et al.2010.Mitochondria beta-cyanoalanine synthase is essential for root hair formation in Arabidopsis thaliana[J].Plant Cell,22(10)3268-3279.
Haralampidis K,Trojanowska M,Osbourn A E.2002.Biosynthesis of triterpenoid saponins in plants[J].Advances in Biochemical Engineering/Biotechnology,75(75):31-49.
Iturbe-Ormaetxe I,Haralampidis K,Papadopoulou K,et al2003.Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus[J].Plant Molecular Biology,51(5):731-743.
Liu Y,Cai Y,Zhao Z,et al.2009.Cloning and functional analysis of a beta-Amyrin synthase gene associated with oleanolic ccid biosynthesis in Gentiana straminea MAXIM[J].Biological&Pharmaceutical Bulletin,32(5):818-824.
Liu Y J,Zhao Y J,Su P,et al.2016.The MVA pathway genes expressions and accumulation of celastrol in Tripterygium wilfordii suspension cells in response to methyl jasmonate treatment[J].Journal of Asian Natural Products Research,18(7):619-628.
Ma X,Xu L,Alberobello A T,et al.2015.Celastrol protects against obesity and metabolic dysfunction through activation of a HSF1-PGC1αtranscriptional axis[J].Cel Metabolism,22(4):695-708.
Poralla K,Hewelt A,Prestwich G D,et al.1994.A specific amino acid repeat in squalene and oxidosqualene cyclases[J].Trends in Biochemical Sciences,19(4):157-158.
Kushiro T,Shibuya M,Masuda K,et al.2000.Mutational studies on triterpene synthases:Engineering lupeol synthase intoβ-Amyrin synthase[J].Journal of the American Chemical Society,122(29):6816-6824.
Wang C,Shi C,Yang X,et al.2014.Celastrol suppresses obesity process via increasing antioxidant capacity and improving lipid metabolism[J].European Journal of Pharmacology,744:52-58.
Yang H,Chen D,Cui Q C,et al.2006.Celastrol,a triterpene extracted from the Chinese"Thunder of God Vine,"is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice[J].Cancer Research,66(9):4758-4765.
Zhu C S,Miao G P,Guo J,et al.2014.Establishment of Tripterygium wilfordii Hook.f.hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine[J].Journal of Microbiology Biotechnology,24(6):823-834.
罗都强,张兴,冯俊涛.2000.杀虫植物雷公藤研究进展[J]西北农林科技大学学报(自然科学版),28(3):84-89(Luo D Q,Zhang X,Feng J T.2000.Research development of an insecticidal plant Tripterygium wilfordii Hook[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry(Natural Science Edition),28(3)84-89.)
梁新华,樊平.2016.雷公藤多苷片对糖尿病肾脏病变的临床疗效系统评价[J].山西医科大学学报,47(8):751-755.(Liang X H,Fan P.2016.Systemic evaluation of clinical effects of Tripterygium glycosides on diabetic nephropathy[J].Journal of Shanxi Medical University47(8):751-755.)
刘佳,付爽,王萌,等.2017.低温胁迫下人参愈伤组织中氧化鲨烯环化酶基因家族的表达特性分析[J].北方园艺,41(17):164-168.(Liu J,Shuang F U,Wang M,et al2017.Effect of chilling stress on expression characteristic of oxidosqualene cyclase(OSC)in callus of Panax ginseng[J].Northern Horticulture,41(17):164-168.)
任献青,鲁静,孟祥乐,等.2013.雷公藤红素药理作用最新研究进展[J].中华中医药杂志,28(9):2679-2682.(Ren X Q,Lu J,Meng X L,et al.2013.Recent progress on pharmacological effects of celastrol[J].China Journal of Traditional Chinese Medicine and Pharmacy,28(9):2679-2682.)
沈湛云,刘春生,王学勇.2009.甘草β-香树酯醇合成酶的编码区克隆与序列分析[J].中国中药杂志,2009,34(19):2438-2440.(Shen Z Y,Liu C S,Wang X Y.2009.Cloning and characterization of open reading frame encodingβ-amyrin synthase in Glycyrrhiza uralensis[J].China Journal of Chinese Materia Medica,34(19):2438-2440.)
夏焱,段宏泉,张铁军,等.2005.雷公藤属药用植物的研究进展[J].中草药,36(7):1093-1096.(Xia Y,Duan H Q,Zhang T J,et al.2005.Advances in studies on medicinal plants of Tripterygium Hook.f.[J].Chinese Traditional and Herbal Drugs,36(7):1093-1096.)
袁凯.2017.雷公藤甲素、雷公藤红素通过调节中性粒细胞活性治疗类风湿关节炎的机制研究[D].博士学位论文,北京中医药大学,导师:徐安龙,pp.18-23.(Yuan K.2017.Triptolide and celastrol treatment of rheumatoid arthritis by regulating neutrophil activity[D].Thesis for Ph.D.,Beijing University of Chinese Medicine,Supervisor:Xu A L,pp.18-23.)
Basyuni M,Oku H,Tsujimoto E,et al.2010.Triterpene synthases from the Okinawan mangrove tribe Rhizophoraceae[J].FEBS Journal,274(19):5028-5042.
Brinker A M,Ma J,Lipsky P E,et al.2007.Medicinal chemistry and pharmacology of genus Tripterygium(Celastraceae)[J].Phytochemistry,38(27):732-766.
Brinker A M,Raskin I.2005.Determination of triptolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reverse phase high-performance liquid chromatography[J].Journal of Chromatography A,1070(1-2):65-70.
Choi D W,Jung J D,Ha Y I,et al.2005.Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites[J].Plant Cell Reports,23(8):557-566.
Chang C H,Chen Y C,Tseng S W,et al.2012.The cysteine703 to isoleucine or histidine mutation of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae generates an iridal-type triterpenoid[J].Biochimie,94(11):2376-2381.
García I,Castellano J M,Vioque B,et al.2010.Mitochondria beta-cyanoalanine synthase is essential for root hair formation in Arabidopsis thaliana[J].Plant Cell,22(10)3268-3279.
Haralampidis K,Trojanowska M,Osbourn A E.2002.Biosynthesis of triterpenoid saponins in plants[J].Advances in Biochemical Engineering/Biotechnology,75(75):31-49.
Iturbe-Ormaetxe I,Haralampidis K,Papadopoulou K,et al2003.Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus[J].Plant Molecular Biology,51(5):731-743.
Liu Y,Cai Y,Zhao Z,et al.2009.Cloning and functional analysis of a beta-Amyrin synthase gene associated with oleanolic ccid biosynthesis in Gentiana straminea MAXIM[J].Biological&Pharmaceutical Bulletin,32(5):818-824.
Liu Y J,Zhao Y J,Su P,et al.2016.The MVA pathway genes expressions and accumulation of celastrol in Tripterygium wilfordii suspension cells in response to methyl jasmonate treatment[J].Journal of Asian Natural Products Research,18(7):619-628.
Ma X,Xu L,Alberobello A T,et al.2015.Celastrol protects against obesity and metabolic dysfunction through activation of a HSF1-PGC1αtranscriptional axis[J].Cel Metabolism,22(4):695-708.
Poralla K,Hewelt A,Prestwich G D,et al.1994.A specific amino acid repeat in squalene and oxidosqualene cyclases[J].Trends in Biochemical Sciences,19(4):157-158.
Kushiro T,Shibuya M,Masuda K,et al.2000.Mutational studies on triterpene synthases:Engineering lupeol synthase intoβ-Amyrin synthase[J].Journal of the American Chemical Society,122(29):6816-6824.
Wang C,Shi C,Yang X,et al.2014.Celastrol suppresses obesity process via increasing antioxidant capacity and improving lipid metabolism[J].European Journal of Pharmacology,744:52-58.
Yang H,Chen D,Cui Q C,et al.2006.Celastrol,a triterpene extracted from the Chinese"Thunder of God Vine,"is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice[J].Cancer Research,66(9):4758-4765.
Zhu C S,Miao G P,Guo J,et al.2014.Establishment of Tripterygium wilfordii Hook.f.hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine[J].Journal of Microbiology Biotechnology,24(6):823-834.