辣木SCoT-PCR反应体系建立和引物筛选
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摘要
SCoT-PCR是一种单引物扩增分子标记方法,具有丰富的多态性,低廉的成本和较好的引物通用性,已在多种植物上加以应用。本研究采用L16(45)正交试验设计,对SCoT-PCR的影响因素进行优化试验,建立了适用于辣木的最佳SCoT-PCR反应体系,并对40条SCoT引物进行筛选。结果表明,25μL最佳反应总体积包含2.0 mmol/L Mg~(2+),0.3 mmol/L d NTPs,1.5 U ExTaq,1.25μmol/L引物,50 ng DNA模板,其中,dNTPs对该体系的影响程度最大。此外,40条SCoT引物共筛选出15条多态性好且适合辣木扩增的引物。该体系的建立为今后利用SCoT分子标记分析辣木种质资源鉴定与多样性、构建指纹图谱和辅助选择育种提供新的技术手段。
SCoT-PCR is a molecular marker method of single primer amplification and is widely used in various plants for its abundant polymorphism, cheap cost and primer universality. The study used the L16(45) orthogonal design to carry out the optimizing experiment of the influence factors of SCoT-PCR. We established the optimum SCoT-PCR reaction system for Moringa oleifera and screened 40 SCoT primers. The result showed that the total volume of the25 μL best reaction contained 2.0 mmol/L Mg2+, 0.3 mmol/L d NTPs, 1.5 U Ex Taq, 1.25 μmol/L primer and 50 ng DNA template, among which d NTPs had the greatest impact on the system. Furthermore, 15 primers with good polymorphism which were fit for Moringa amplification were screened from the 40 SCoT primers. The establishment of this system might provide new technological means for future use of SCoT molecular markers to analyze the identification and diversity of Moringa oleifera germplasm resources and to build fingerprint and marker assisted selection.
SCoT-PCR is a molecular marker method of single primer amplification and is widely used in various plants for its abundant polymorphism, cheap cost and primer universality. The study used the L16(45) orthogonal design to carry out the optimizing experiment of the influence factors of SCoT-PCR. We established the optimum SCoT-PCR reaction system for Moringa oleifera and screened 40 SCoT primers. The result showed that the total volume of the25 μL best reaction contained 2.0 mmol/L Mg2+, 0.3 mmol/L d NTPs, 1.5 U Ex Taq, 1.25 μmol/L primer and 50 ng DNA template, among which d NTPs had the greatest impact on the system. Furthermore, 15 primers with good polymorphism which were fit for Moringa amplification were screened from the 40 SCoT primers. The establishment of this system might provide new technological means for future use of SCoT molecular markers to analyze the identification and diversity of Moringa oleifera germplasm resources and to build fingerprint and marker assisted selection.
引文
Collard B.C.Y.,and M ackill D.J.,2009,Start codon targeted(SCOT)polymorphism:A simple,novel DNA marker technique for generating gene-targeted markers in plants,Plant Molecular Biology Reporter,27(1):86-93
Han C.,Li M.,Zhong R.,Yun Y.,and Yang S.C.,2008,Study on Moringa plants by ISSR marker,Xiamen Daxue Xuebao(Journal of Xiamen University),47(2):177-180(韩闯,李敏,钟然,云叶,杨盛昌,2008,辣木植物ISSR分子标记的初步研究,厦门大学学报,47(2):177-180)
Han G.H.,Xiang S.Q.,Wang W.X.,Jia Z.G.,Hong Q.B.,and Liang G.L.,2011,Establishment and application of SCo Tmolecular marker system for citrus,Yuanyi Xuebao(Acta Horticulturae Sinica),38(7):1243-1250(韩国辉,向素琼,汪卫星,贾志刚,洪棋斌,梁国鲁,2011,柑橘SCo T分子标记技术体系的建立及其在遗传分析中的应用,园艺学报,38(7):1243-1250)
He Z.W.,Liu Y.S.,Chen L.H.,and Cao M.H.,1998,Orthogonal design-direct analysis for PCR optimization,Hunan Yike Daxue Xuebao(Bulletin of Hunan Medical Universit),23(4):403-404(何正文,刘运生,陈立华,曹美鸿,1998,正交设计直观分析法优化PCR条件,湖南医科大学学报,23(4):403-404)
Hou X.G.,Wang J.,Jia T.,Zhang Q.Y.,Hou J.,and Li J.Y.,2011,Orthogonal optimization of SCo T-PCR system and primer screening of tree peony,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),26(5):92-96(侯小改,王娟,贾甜,张钰乾,侯娟,李嘉钰,2011,牡丹SCo T分子标记正交优化及引物筛选,华北农学报,26(5):92-96)
Li H.B.,Ren A.X.,Ma C.J.,and Wang Y.Z.,2009a,Construction of RAPD optimal reaction system in Moringa oleifera Lamarch,Anhui Nongye Kexue(Journal of Anhui Agriculture Science),37(2):504-505(李海渤,任安祥,马崇坚,王玉珍,2009a,辣木RAPD最佳反应体系的构建,安徽农业科学,37(2):504-505)
Li H.B.,Tang Z.M.,Ren A.X.,Ma C.J.,and Wang Y.Z.,2009b,Genetic diversity analysis of Moringa oleifera based on RAPD markers,Jiangsu Nongye Kexue(Jiangsu Agriculture Science),(1):49-51(李海渤,唐志明,任安祥,马崇坚,王玉珍,2009b,用RAPD标记分析辣木的遗传多样性,江苏农业科学,(1):49-51)
Li N.,Yang T.,Yang S.B.,Tang Y.P.,Wang B.K.,Wang Q.,Pai T.G.L.,and Yu Q.H.,2015,Optimization of SCo T-PCR system and primers screening of dried-usage chili,Nanfang Nongye Xuebao(Journal of Southern Agriculture),46(4):544-549(李宁,杨涛,杨生保,唐亚萍,王柏柯,王强,帕提古丽,余庆辉,2015,制干辣椒SCo T-PCR体系优化及引物筛选,南方农业学报,46(4):544-549)
Liu J.X.,Xiong F.Q.,Liu X.,Liu L.M.,Luo L.,Liu H.J.,Yu K.X.,Dan M.,Lu M.M.,He Y.B.,and Li S.,2016,Molecular identification of fruit sugarcane badila and its bud sports with combination of SCo T,BPS and URP markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(1):190-197(刘俊仙,熊发前,刘欣,刘丽敏,罗丽,刘红坚,余坤兴,淡明,卢曼曼,何毅波,李松,2016,果蔗拔地拉及其芽变株系的SCo T、BPS和URP联合分子鉴定,基因组学与应用生物学,36(1):190-197)
Luo C.,He X.H.,Chen H.,Ou S.J.,and Gao M.P.,2010,Analysis of diversity and relationships among mango cultivars using start codon targeted(SCOT)markers,Biochemical Systematics and Ecology,38(6):1176-1184
Su Y.C.,Ling H.,Wang H.B.,Que Y.X.,Wu Q.B.,Chen S.S.,and Xu L.P.,2012,Optimization of SCo T-PCR reaction system,and screening and utilization of polymorphic primers in Sugarcane,Yingyong Yu Huanjin Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),18(5):810-818(苏亚春,凌辉,王恒波,阙友雄,吴期滨,陈珊珊,许莉萍,2012,甘蔗SCo T-PCR反应体系优化与多态性引物筛选及应用,应用与环境生物学报,18(5):810-818)
Wu J.M.,Li Y.R.,Wang A.Q.,Yang L.,and Yang L.T.,2010,Differential expression of genes in gibberellin-induced stalk elongation of sugarcane analyzed with c DNA-Sco T,Zuowu Xuebao(Acta Agronomica Sinica),36(11):1883-1890(吴建明,李杨瑞,王爱勤,杨柳,杨丽涛,2010,赤霉素诱导甘蔗节间伸长基因的c DNA-SCo T差异表达分析,作物学报,36(11):l883-1890)
Xiong F.Q.,Jiang J.,Zhong R.C.,Han Z.Q.,He L.Q.,Li Z.,Zhuang W.J.,and Tang R.H.,2010,Application of SCo Tmolecular marker in Genus arachis,Zuowu Xuebao(Acta Agronomica Sinica),36(12):2055-2061(熊发前,蒋菁,钟瑞春,韩柱强,贺梁琼,李忠,庄伟建,唐荣华,2010,目标起始密码子多态性(SCOT)分子标记技术在花生属中的应用,作物学报,36(12):2055-2061)
Xiong F.Q.,Tang R.H.,Chen Z.L.,Pan L.H.,and Zhuang W.J.,2009,SCo T:a novel gene targeted marker technique based on the translation start codon,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(3):635-638(熊发前,唐荣华,陈忠良,潘玲华,庄伟建,2009,目标起始密码子多态性(SCOT):一种基于翻译起始位点目的基因标记技术,分子植物育种,7(3):635-638)
Yang J.,Wang P.,and Shi L.,2016,Optimization for SCo T-PCRsystem and primer selection of seed-used watermelon,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),25(6):889-896(杨静,王萍,石磊,2016,籽用西瓜SCo T-PCR反应体系优化及引物筛选,西北农业学报,25(6):889-896)
Yang X.Y.,Cai Y.B.,Guo L.F.,Zhou Q.G.,Chen H.J.,and Sun G.M.,2012,Establishment of SCo T amplification system and primer selection for Carica papaya,Redai Yaredai Zhiwu Xuebao(Journal of Tropical and Subtropical Botany),20(6):578-584(杨祥燕,蔡元保,郭凌飞,周全光,陈豪军,孙光明,2012,番木瓜SCo T反应体系建立及引物筛选,热带亚热带植物学报,20(6):578-584)
Zhao R.Q.,Gao Y.H.,Zhang X.L.,and Si J.P.,2012,Establishment and optimizatio n of SCo T-PCR reaction system for Dendrobium officinale,Henongxue Bao(Journal of Nuclear Agricultural Sciences),26(4):648-655(赵瑞强,高燕会,章晓玲,斯金平,2012,铁皮石斛SCo T-PCR反应体系构建及优化,核农学报,26(4):648-655)
Zheng S.L.,Shao J.H.,Zhang J.L.,Gao X.,Tian Y.,Zhang G.H.,Yang S.C.,and Ma C.H.,2016,Genetic diversity analysis of Yunnan Moringa oleifera Lam,with AFLP markers,Yunnan Nongye Daxue Xubao(Journal of Yunnan University),31(5):850 -855(郑硕理,邵建辉,张敬丽,高熹,田洋,张广辉,杨生超,马春花,2016,云南辣木资源遗传多样性的AFLP分析,云南农业大学学报,31(5):850-855)
Han C.,Li M.,Zhong R.,Yun Y.,and Yang S.C.,2008,Study on Moringa plants by ISSR marker,Xiamen Daxue Xuebao(Journal of Xiamen University),47(2):177-180(韩闯,李敏,钟然,云叶,杨盛昌,2008,辣木植物ISSR分子标记的初步研究,厦门大学学报,47(2):177-180)
Han G.H.,Xiang S.Q.,Wang W.X.,Jia Z.G.,Hong Q.B.,and Liang G.L.,2011,Establishment and application of SCo Tmolecular marker system for citrus,Yuanyi Xuebao(Acta Horticulturae Sinica),38(7):1243-1250(韩国辉,向素琼,汪卫星,贾志刚,洪棋斌,梁国鲁,2011,柑橘SCo T分子标记技术体系的建立及其在遗传分析中的应用,园艺学报,38(7):1243-1250)
He Z.W.,Liu Y.S.,Chen L.H.,and Cao M.H.,1998,Orthogonal design-direct analysis for PCR optimization,Hunan Yike Daxue Xuebao(Bulletin of Hunan Medical Universit),23(4):403-404(何正文,刘运生,陈立华,曹美鸿,1998,正交设计直观分析法优化PCR条件,湖南医科大学学报,23(4):403-404)
Hou X.G.,Wang J.,Jia T.,Zhang Q.Y.,Hou J.,and Li J.Y.,2011,Orthogonal optimization of SCo T-PCR system and primer screening of tree peony,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),26(5):92-96(侯小改,王娟,贾甜,张钰乾,侯娟,李嘉钰,2011,牡丹SCo T分子标记正交优化及引物筛选,华北农学报,26(5):92-96)
Li H.B.,Ren A.X.,Ma C.J.,and Wang Y.Z.,2009a,Construction of RAPD optimal reaction system in Moringa oleifera Lamarch,Anhui Nongye Kexue(Journal of Anhui Agriculture Science),37(2):504-505(李海渤,任安祥,马崇坚,王玉珍,2009a,辣木RAPD最佳反应体系的构建,安徽农业科学,37(2):504-505)
Li H.B.,Tang Z.M.,Ren A.X.,Ma C.J.,and Wang Y.Z.,2009b,Genetic diversity analysis of Moringa oleifera based on RAPD markers,Jiangsu Nongye Kexue(Jiangsu Agriculture Science),(1):49-51(李海渤,唐志明,任安祥,马崇坚,王玉珍,2009b,用RAPD标记分析辣木的遗传多样性,江苏农业科学,(1):49-51)
Li N.,Yang T.,Yang S.B.,Tang Y.P.,Wang B.K.,Wang Q.,Pai T.G.L.,and Yu Q.H.,2015,Optimization of SCo T-PCR system and primers screening of dried-usage chili,Nanfang Nongye Xuebao(Journal of Southern Agriculture),46(4):544-549(李宁,杨涛,杨生保,唐亚萍,王柏柯,王强,帕提古丽,余庆辉,2015,制干辣椒SCo T-PCR体系优化及引物筛选,南方农业学报,46(4):544-549)
Liu J.X.,Xiong F.Q.,Liu X.,Liu L.M.,Luo L.,Liu H.J.,Yu K.X.,Dan M.,Lu M.M.,He Y.B.,and Li S.,2016,Molecular identification of fruit sugarcane badila and its bud sports with combination of SCo T,BPS and URP markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(1):190-197(刘俊仙,熊发前,刘欣,刘丽敏,罗丽,刘红坚,余坤兴,淡明,卢曼曼,何毅波,李松,2016,果蔗拔地拉及其芽变株系的SCo T、BPS和URP联合分子鉴定,基因组学与应用生物学,36(1):190-197)
Luo C.,He X.H.,Chen H.,Ou S.J.,and Gao M.P.,2010,Analysis of diversity and relationships among mango cultivars using start codon targeted(SCOT)markers,Biochemical Systematics and Ecology,38(6):1176-1184
Su Y.C.,Ling H.,Wang H.B.,Que Y.X.,Wu Q.B.,Chen S.S.,and Xu L.P.,2012,Optimization of SCo T-PCR reaction system,and screening and utilization of polymorphic primers in Sugarcane,Yingyong Yu Huanjin Shengwu Xuebao(Chinese Journal of Applied and Environmental Biology),18(5):810-818(苏亚春,凌辉,王恒波,阙友雄,吴期滨,陈珊珊,许莉萍,2012,甘蔗SCo T-PCR反应体系优化与多态性引物筛选及应用,应用与环境生物学报,18(5):810-818)
Wu J.M.,Li Y.R.,Wang A.Q.,Yang L.,and Yang L.T.,2010,Differential expression of genes in gibberellin-induced stalk elongation of sugarcane analyzed with c DNA-Sco T,Zuowu Xuebao(Acta Agronomica Sinica),36(11):1883-1890(吴建明,李杨瑞,王爱勤,杨柳,杨丽涛,2010,赤霉素诱导甘蔗节间伸长基因的c DNA-SCo T差异表达分析,作物学报,36(11):l883-1890)
Xiong F.Q.,Jiang J.,Zhong R.C.,Han Z.Q.,He L.Q.,Li Z.,Zhuang W.J.,and Tang R.H.,2010,Application of SCo Tmolecular marker in Genus arachis,Zuowu Xuebao(Acta Agronomica Sinica),36(12):2055-2061(熊发前,蒋菁,钟瑞春,韩柱强,贺梁琼,李忠,庄伟建,唐荣华,2010,目标起始密码子多态性(SCOT)分子标记技术在花生属中的应用,作物学报,36(12):2055-2061)
Xiong F.Q.,Tang R.H.,Chen Z.L.,Pan L.H.,and Zhuang W.J.,2009,SCo T:a novel gene targeted marker technique based on the translation start codon,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(3):635-638(熊发前,唐荣华,陈忠良,潘玲华,庄伟建,2009,目标起始密码子多态性(SCOT):一种基于翻译起始位点目的基因标记技术,分子植物育种,7(3):635-638)
Yang J.,Wang P.,and Shi L.,2016,Optimization for SCo T-PCRsystem and primer selection of seed-used watermelon,Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),25(6):889-896(杨静,王萍,石磊,2016,籽用西瓜SCo T-PCR反应体系优化及引物筛选,西北农业学报,25(6):889-896)
Yang X.Y.,Cai Y.B.,Guo L.F.,Zhou Q.G.,Chen H.J.,and Sun G.M.,2012,Establishment of SCo T amplification system and primer selection for Carica papaya,Redai Yaredai Zhiwu Xuebao(Journal of Tropical and Subtropical Botany),20(6):578-584(杨祥燕,蔡元保,郭凌飞,周全光,陈豪军,孙光明,2012,番木瓜SCo T反应体系建立及引物筛选,热带亚热带植物学报,20(6):578-584)
Zhao R.Q.,Gao Y.H.,Zhang X.L.,and Si J.P.,2012,Establishment and optimizatio n of SCo T-PCR reaction system for Dendrobium officinale,Henongxue Bao(Journal of Nuclear Agricultural Sciences),26(4):648-655(赵瑞强,高燕会,章晓玲,斯金平,2012,铁皮石斛SCo T-PCR反应体系构建及优化,核农学报,26(4):648-655)
Zheng S.L.,Shao J.H.,Zhang J.L.,Gao X.,Tian Y.,Zhang G.H.,Yang S.C.,and Ma C.H.,2016,Genetic diversity analysis of Yunnan Moringa oleifera Lam,with AFLP markers,Yunnan Nongye Daxue Xubao(Journal of Yunnan University),31(5):850 -855(郑硕理,邵建辉,张敬丽,高熹,田洋,张广辉,杨生超,马春花,2016,云南辣木资源遗传多样性的AFLP分析,云南农业大学学报,31(5):850-855)