丹参KSL基因多态性对其编码酶催化反应的影响
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摘要
目的分析全国不同产区丹参KSL基因的变异类型,初步探究丹参KSL基因多态性对其编码酶催化效率的影响。方法设计特异性引物,同源克隆河南、山东、四川、湖北、山西、内蒙古、河北产区丹参KSL基因全长,比较变异类型,选取突变样品构建原核表达体系,进行体外诱导表达;通过气相色谱-质谱(GS-MS)技术检测该基因酶催化产物,进一步分析丹参KSL基因多态性对蛋白酶催化效率的影响。结果丹参KSL基因全长1 788 bp,共编码595个氨基酸,不同产区的丹参KSL基因存在基因多态性,并能够引起同义和非同义突变。丹参KSL不同基因型中催化效率明显不同,山西产区的催化效率最高,内蒙古产区的催化效率最低。其中,产自山西、湖北、山东、四川的丹参均包含1 047 bp处A型变异,其催化效率明显高于不包含A型变异的河南、河北、内蒙古3个产区的丹参的催化效率。结论不同产区丹参KSL基因存在多态性,由基因多态性引起的非同义突变进一步导致氨基酸序列的变化,最终使编码酶的催化效率产生差异。以丹参酮类成分合成途径中关键酶基因的多态性作为切入点,为丹参酮类成分积累机制的揭示及丹参优良种质的选育提供进一步的参考。
Objective To analyze the variation types kaunene synthase-like(KSL)gene in Danshen(Salvia Root,Radix Salviae Miltiorrhizae)from different producing areas in China,and investigate primarily the influence of KSL gene polymorphism on enzyme-catalyzed reaction of its codases.Methods Specific primers were designed,and full-length KSL genes of Danshen from different producing areas(Henan,Shandong,Sichuan,Hubei,Shanxi,Neimonggu,Hebei)in China were homologously cloned.The variation types were compared and mutation samples were selected to establish a prokaryotic expression system and inducible expression in vitro.The enzyme-catalyzed products of KSL gene were detected by using gas chromatography-mass spectrometer(GS-MS),and influence of KSL gene polymorphism of Danshen on protease catalytic efficiency was further analyzed.Results The full-length KSL gene was 1 788 bp,and there were totally 595 amino acid residues encoded.The sequencing results showed that there were polymorphisms in KSL gene of Danshen from different producing areas in China,which could cause synonymous and non-synonymous mutations.The catalytic efficiency of different genotypes of KSL was significantly different,which was the highest in Shanxi and the lowest in Inner Mongolia.The KSL gene containing A-type variation mutated at 1 047 bp in Danshen from4 producing areas including Shanxi,Hubei,Shandong,and Sichuan,and the catalytic efficiency of which was significantly higher than those containing no Atype variation in Danshen from4 producing areas including Henan,Hebei and Inner Mongolia.Conclusion KSL genes have gene polymorphisms indeed in Danshen from different producing areas.The non-synonymous mutations induced by polymorphisms can further lead to changes of amino acid sequences and finally resulting in difference in the catalytic efficiency of encoded enzymes.The polymorphisms of key enzyme genes in the synthesis pathway of tanshinones can be taken as a starting point to provide further reference for revealing the accumulation mechanism of tanshinones and breeding the excellent germplasm of Danshen.
Objective To analyze the variation types kaunene synthase-like(KSL)gene in Danshen(Salvia Root,Radix Salviae Miltiorrhizae)from different producing areas in China,and investigate primarily the influence of KSL gene polymorphism on enzyme-catalyzed reaction of its codases.Methods Specific primers were designed,and full-length KSL genes of Danshen from different producing areas(Henan,Shandong,Sichuan,Hubei,Shanxi,Neimonggu,Hebei)in China were homologously cloned.The variation types were compared and mutation samples were selected to establish a prokaryotic expression system and inducible expression in vitro.The enzyme-catalyzed products of KSL gene were detected by using gas chromatography-mass spectrometer(GS-MS),and influence of KSL gene polymorphism of Danshen on protease catalytic efficiency was further analyzed.Results The full-length KSL gene was 1 788 bp,and there were totally 595 amino acid residues encoded.The sequencing results showed that there were polymorphisms in KSL gene of Danshen from different producing areas in China,which could cause synonymous and non-synonymous mutations.The catalytic efficiency of different genotypes of KSL was significantly different,which was the highest in Shanxi and the lowest in Inner Mongolia.The KSL gene containing A-type variation mutated at 1 047 bp in Danshen from4 producing areas including Shanxi,Hubei,Shandong,and Sichuan,and the catalytic efficiency of which was significantly higher than those containing no Atype variation in Danshen from4 producing areas including Henan,Hebei and Inner Mongolia.Conclusion KSL genes have gene polymorphisms indeed in Danshen from different producing areas.The non-synonymous mutations induced by polymorphisms can further lead to changes of amino acid sequences and finally resulting in difference in the catalytic efficiency of encoded enzymes.The polymorphisms of key enzyme genes in the synthesis pathway of tanshinones can be taken as a starting point to provide further reference for revealing the accumulation mechanism of tanshinones and breeding the excellent germplasm of Danshen.
引文
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[3]周晓希,孔羽,崔浪军,等.RP-HPLC-DAD同时测定丹参及丹参片中6种水溶性成分的含量[J].中药材,2014,37(2):337-339.Zhou XX,Kong Y,Cui LJ,et al.Simultaneous determination of six water-soluble components in Danshen and Danshen tablets by RP-HPLC-DAD[J].Journal of Chinese Medicinal Materials,2014,37(2):337-339.
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[6]葛宇清,杨波,程汝滨,等.隐丹参酮对K562细胞凋亡的影响及其机制[J].中草药,2013,44(22):3188-3194.Ge YQ,Yang B,Cheng RB,et al.Effect of cryptotanshinone on apoptosis in K562 cell and its mechanism[J].Chinese Traditional and Herbal Drugs,2013,44(22):3188-3194.
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[8]刘洪伟,王帅,王强,等.甜菜树贝壳杉烯合酶基因的克隆与原核表达[J].食品与生物技术学报,2017,36(5):479-485.Liu HW,Wang S,Wang Q,et al.Cloning and expression of the Ent-kaurene synthase gene from Yunnanopilia longistaminata[J].Journal of Food Science and Biotechnology,2017,36(5):479-485.
[9]程飞飞,欧春青,姜淑苓,等.梨内根-贝壳杉烯合酶基因克隆及表达分析[J].沈阳农业大学学报,2011,42(6):677-682.Cheng FF,Ou CQ,Jiang SL,et al.Cloning and expression analysis of Ent-kaurene synthase gene in pear[J].Journal of Shenyang Agricultural University,2011,42(6):677-682.
[10]Jin BL,Cui GH,Guo J,et al.Functional diversification of kaurene synthase-like genes in Isodon rubescens[J].Plant Physiol,2017,174(2):943-955.
[11]Yamamura Y,Kurosaki F,Lee JB.Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis[J/OL].Scientific Reports,2017(7):43311[2017-03-07].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339715/.
[12]Ming QL,Su CY,Zheng CJ,et al.Elicitors from the endophytic fungus Trichoderma atroviride promote Salvia miltiorrhiza hairy root growth and tanshinone biosynthesis[J].Journal of Experimental Botany,2013,64(18):5687-5694.
[13]呙未.甘草SQS1基因多态性及其对产物积累的影响研究[D].北京:北京中医药大学,2010.Guo W.Polymorphism of SQS1 gene in Glycyrrhiza and its effect on product accumulation[D].Beijing:Beijing University of Chinese Medicine,2010.
[14]许巧仙.甘草HMGR基因多态性及其对产物MVA积累的影响研究[D].北京:北京中医药大学,2010.Xu QX.Polymorphism of HMGR gene in Liquorice and its effect on MVA accumulation[D].Beijing:Beijing University of Chinese Medicine,2010.
[15]Zhang JX,Zhou LB,Zheng XY,et al.Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots[J].Plant Cell Reports,2017,36(8):1297-1309.
[16]Cui GH,Feng H,Li WY,et al.Cloning and polymorphism analysis of SmERF in Salvia miltiorrhiza[J].Acta Pharmaceutica Sinica,2010,45(9):1188-1193.
[17]沈湛云,刘春生,黄建梅,等.甘草β-香树酯醇合成酶的多态性对其催化效率的影响研究[J].中国中药杂志,2010,35(22):2941-2944.Shen ZY,Liu CS,Huang JM,et al.Impacting of polymorphism ofβ-amyrin synthase from Glycyrrhiza uralensis on its catalytic efficiency[J].China Journal of Chinese Materia Medica,2010,35(22):2941-2944.
[18]李贝宁,周晓丽,黄璐琦,等.不同产地丹参功能基因表达水平对丹参酮类成分积累的影响[J].中国中药杂志,2011,36(24):3406-3409.Li BN,Zhou XL,Huang LQ,et al.Correlation between expression level of functional genes and tanshinones'accumulation in Salvia miltiorrhiza from different areas[J].China Journal of Chinese Materia Medica,2011,36(24):3406-3409.
[19]Wang CH,Zheng LP,Tian H,et al.Synergistic effects of ultraviolet-B and methyl jasmonate on tanshinone biosynthesis in Salvia miltiorrhiza hairy roots[J].J Photochem Photobiol B:Biology,2016,159:93-100.
[20]Bai ZQ,Liu JL,Zhang CL,et al.Coding single nucleotide polymorphisms and SmCPS1 and SmKSL1 subcellular localization are associated with tanshinone biosynthesis in Salvia miltiorrhiza Bunge roots[J/OL].Acta Physiologiae Plantarum,2018,40:6[2017-12-04].https://link.springer.com/artiale/10.1007%2Fs11738-017-2563-x.
[21]刘颖,张宁,王学勇,等.甘草鲨烯合酶基因多态性对其编码酶催化效率影响的研究[J].中国中药杂志,2012,37(24):3777-3783.Liu Y,Zhang N,Wang XY,et al.Researches on influence of squalene synthase gene polymorphism on catalytic efficiency of its encode enzyme in Glycyrrhiza uralensis[J].China Journal of Chinese Materia Medica,2012,37(24):3777-3783.
[2]Su CY,Ming QL,Rahman K,et al.Salvia miltiorrhiza:traditional medicinal uses,chemistry,and pharmacology[J].Chin J Nat Med,2015,13(3):163-182.
[3]周晓希,孔羽,崔浪军,等.RP-HPLC-DAD同时测定丹参及丹参片中6种水溶性成分的含量[J].中药材,2014,37(2):337-339.Zhou XX,Kong Y,Cui LJ,et al.Simultaneous determination of six water-soluble components in Danshen and Danshen tablets by RP-HPLC-DAD[J].Journal of Chinese Medicinal Materials,2014,37(2):337-339.
[4]Ma PD,Liu JL,Zhang CL,et al.Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza Bunge[J]Appl Biochem Biotechnol,2013,170(6):1253-1262.
[5]郑娟,沈成英,庞建云,等.丹参酮Ⅱ_A纳米结构脂质载体的体外评价及其对HaCaT细胞增殖的影响[J].中草药,2016,47(24):4340-4344.Zheng J,Shen CY,Pang JY,et al.Evaluation and effects on HaCaT cells in vitro of tanshinone IIA-loaded nanostructured lipid carrier[J].Chinese Traditional and Herbal Drugs,2016,47(24):4340-4344.
[6]葛宇清,杨波,程汝滨,等.隐丹参酮对K562细胞凋亡的影响及其机制[J].中草药,2013,44(22):3188-3194.Ge YQ,Yang B,Cheng RB,et al.Effect of cryptotanshinone on apoptosis in K562 cell and its mechanism[J].Chinese Traditional and Herbal Drugs,2013,44(22):3188-3194.
[7]高伟.丹参酮类化合物生物合成相关酶基因克隆及功能研究[D].北京:中国中医科学院,2008.Gao W.Cloning and functional analysis on genes related to biosynthesis of tanshinone compounds[D].Beijing:China Academy of Chinese Medical Sciences,2008.
[8]刘洪伟,王帅,王强,等.甜菜树贝壳杉烯合酶基因的克隆与原核表达[J].食品与生物技术学报,2017,36(5):479-485.Liu HW,Wang S,Wang Q,et al.Cloning and expression of the Ent-kaurene synthase gene from Yunnanopilia longistaminata[J].Journal of Food Science and Biotechnology,2017,36(5):479-485.
[9]程飞飞,欧春青,姜淑苓,等.梨内根-贝壳杉烯合酶基因克隆及表达分析[J].沈阳农业大学学报,2011,42(6):677-682.Cheng FF,Ou CQ,Jiang SL,et al.Cloning and expression analysis of Ent-kaurene synthase gene in pear[J].Journal of Shenyang Agricultural University,2011,42(6):677-682.
[10]Jin BL,Cui GH,Guo J,et al.Functional diversification of kaurene synthase-like genes in Isodon rubescens[J].Plant Physiol,2017,174(2):943-955.
[11]Yamamura Y,Kurosaki F,Lee JB.Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis[J/OL].Scientific Reports,2017(7):43311[2017-03-07].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339715/.
[12]Ming QL,Su CY,Zheng CJ,et al.Elicitors from the endophytic fungus Trichoderma atroviride promote Salvia miltiorrhiza hairy root growth and tanshinone biosynthesis[J].Journal of Experimental Botany,2013,64(18):5687-5694.
[13]呙未.甘草SQS1基因多态性及其对产物积累的影响研究[D].北京:北京中医药大学,2010.Guo W.Polymorphism of SQS1 gene in Glycyrrhiza and its effect on product accumulation[D].Beijing:Beijing University of Chinese Medicine,2010.
[14]许巧仙.甘草HMGR基因多态性及其对产物MVA积累的影响研究[D].北京:北京中医药大学,2010.Xu QX.Polymorphism of HMGR gene in Liquorice and its effect on MVA accumulation[D].Beijing:Beijing University of Chinese Medicine,2010.
[15]Zhang JX,Zhou LB,Zheng XY,et al.Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots[J].Plant Cell Reports,2017,36(8):1297-1309.
[16]Cui GH,Feng H,Li WY,et al.Cloning and polymorphism analysis of SmERF in Salvia miltiorrhiza[J].Acta Pharmaceutica Sinica,2010,45(9):1188-1193.
[17]沈湛云,刘春生,黄建梅,等.甘草β-香树酯醇合成酶的多态性对其催化效率的影响研究[J].中国中药杂志,2010,35(22):2941-2944.Shen ZY,Liu CS,Huang JM,et al.Impacting of polymorphism ofβ-amyrin synthase from Glycyrrhiza uralensis on its catalytic efficiency[J].China Journal of Chinese Materia Medica,2010,35(22):2941-2944.
[18]李贝宁,周晓丽,黄璐琦,等.不同产地丹参功能基因表达水平对丹参酮类成分积累的影响[J].中国中药杂志,2011,36(24):3406-3409.Li BN,Zhou XL,Huang LQ,et al.Correlation between expression level of functional genes and tanshinones'accumulation in Salvia miltiorrhiza from different areas[J].China Journal of Chinese Materia Medica,2011,36(24):3406-3409.
[19]Wang CH,Zheng LP,Tian H,et al.Synergistic effects of ultraviolet-B and methyl jasmonate on tanshinone biosynthesis in Salvia miltiorrhiza hairy roots[J].J Photochem Photobiol B:Biology,2016,159:93-100.
[20]Bai ZQ,Liu JL,Zhang CL,et al.Coding single nucleotide polymorphisms and SmCPS1 and SmKSL1 subcellular localization are associated with tanshinone biosynthesis in Salvia miltiorrhiza Bunge roots[J/OL].Acta Physiologiae Plantarum,2018,40:6[2017-12-04].https://link.springer.com/artiale/10.1007%2Fs11738-017-2563-x.
[21]刘颖,张宁,王学勇,等.甘草鲨烯合酶基因多态性对其编码酶催化效率影响的研究[J].中国中药杂志,2012,37(24):3777-3783.Liu Y,Zhang N,Wang XY,et al.Researches on influence of squalene synthase gene polymorphism on catalytic efficiency of its encode enzyme in Glycyrrhiza uralensis[J].China Journal of Chinese Materia Medica,2012,37(24):3777-3783.