寒地稗草ISSR-PCR反应体系的正交优化
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摘要
本研究以寒地稗草为实验材料,采用正交试验方法对影响ISSR-PCR反应体系的4个因素进行优化。研究结果表明,寒地稗草ISSR-PCR的最佳反应体系(20μL)为:10×PCR Buffer (含Mg2+) 2μL,dNTPs浓度为0.3 mmol/L,引物浓度为0.2μmol/L,Taq DNA聚合酶为0.75 U,模板DNA含量为20 ng。并确定了引物UBC888的最佳退火温度为51.9℃。利用该反应体系对15份寒地稗草的基因组进行扩增,结果证明优化后的ISSR反应体系稳定可靠,可为稗草种质资源的遗传多样性研究提供理论基础。
Taking Echinochloa crusgalli from cold areas as test materials, the orthogonal design was applied to optimize four main factors which had an influence on ISSR-PCR system. The results showed that the optimum ISSR-PCR reaction system(20 μL) was 2 μL 10×PCR Buffer(Mg2+plus), 0.3 mmol/L dNTPs, 0.2 μmol/L primer,0.75 U Taq DNA polymerase and 20 ng template DNA. And the best annealing temperature of primer UBC888 was determined at 51.9℃. Using this reaction system to amplify the genomes of 15 species of Echinochloa crusgalli from cold areas, the results showed that the optimized ISSR reaction system was stable and reliable,which could settle favorable basis for the study on genetic diversity of Echinochloa crusgalli germplasm resources.
Taking Echinochloa crusgalli from cold areas as test materials, the orthogonal design was applied to optimize four main factors which had an influence on ISSR-PCR system. The results showed that the optimum ISSR-PCR reaction system(20 μL) was 2 μL 10×PCR Buffer(Mg2+plus), 0.3 mmol/L dNTPs, 0.2 μmol/L primer,0.75 U Taq DNA polymerase and 20 ng template DNA. And the best annealing temperature of primer UBC888 was determined at 51.9℃. Using this reaction system to amplify the genomes of 15 species of Echinochloa crusgalli from cold areas, the results showed that the optimized ISSR reaction system was stable and reliable,which could settle favorable basis for the study on genetic diversity of Echinochloa crusgalli germplasm resources.
引文
Feng D.,Ning D.L.,Chen S.Y.,Li Y.J.,Zhang Y.L.,Wu T.,and Chen H.Y.,2014,Optimization of ISSR-PCR reaction system of anise(Illicium verum Hook.F.)based on orthogonal design,Shengwu Jishu Tongbao(Biotechnology Bulletin),(8):65-69(冯丹,宁德鲁,陈少瑜,李勇杰,张艳丽,吴涛,陈海云,2014,正交试验优化八角ISSR-PCR反应体系,生物技术通报,(8):65-69)
Huang X.Y.,Tang H.,Kong D.X.,Wang M.L.,Shi Y.C.,and Zou R.,2013,Establishment and optimization of ISSR-PCRreaction conditions in Rubus suavissimus S.Lee,Jiyinzuzue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(4):526-532(黄夕洋,唐辉,孔德鑫,王满莲,史艳财,邹蓉,2013,广西甜茶ISSR-PCR反应条件的建立与优化,基因组学与应用生物学,32(4):526-532)
He Z.W.,Liu Y.S.,Chen L.H.,Cao M.H.,and Xia J.H.,1998,Orthogonal design-direct analysis for PCR optimization,Hunan Yike Daxue Xuebao(Bulletin of Hunan Medical University),23(4):403-404(何正文,刘运生,陈立华,曹美鸿,夏家辉,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)
Hu Y.P.,Wang L.,Bao R.,Shi L.,Xu R.H.,and Li Y.,2014,Optimizing ISSR-PCR system of Carex atrofusca using orthogonal design,Caodi Xuebao(Acta Agrestia Sinica),22(4):911-914,918(胡延萍,王莉,包蕊,石琳,旭荣花,李毅,2014,正交设计优化黑褐苔草ISSR-PCR反应体系,草地学报,22(4):911-914,918)
Liu D.H.,Lu Y.L.,Lou Y.X.,and Guo S.L.,2016,Optimization of ISSR-PCR reaction system of Echinochloa by orthogonal design,Shanghai Nongye Xuebao(Acta Agriculturae Shanghai),32(4):17-21(刘德好,陆永良,娄玉霞,郭水良,2016,正交试验法优化稗属植物ISSR-PCR反应体系,上海农业学报,32(4):17-21)
Li R.H.,Xia Y.S.,Liu S.Z.,Sun L.L.,Guo P.G.,Miao S.Y.,and Chen J.H.,2009,CTAB-improved method of DNA extractionin plant,Shiyanshi Yanjiu Yu Tansuo(Research and Explorationin Laboratory),28(9):14-16(李荣华,夏岩石,刘顺枝,孙莉丽,郭培国,缪绅裕,陈健辉,2009,改进的CTAB提取植物DNA方法,实验室研究与探索,28(9):14-16)
Jing Q.Y.,Jiao Z.Z.,Liu L.K.,Shi H.,Feng L.,Li W.,and Liu Y.G.,2014,The resistance of barnyardgrass and amaranthus retroflexus to four common used herbicides in corn fields in Heilongjiang province,Zuowu Xuebao(Crops),(5):128-132(井秋月,焦梓洲,刘兰坤,施慧,冯蕾,李威,刘亚光,2014,黑龙江省玉米田稗草与反枝苋对四种常用除草剂的抗药性测定,作物杂志,(5):128-132)
Qu Y.Q.,Xu W.,Shen L.,Chen J.C.,He T.Y.,Wei J.W.,Rong J.D.,and Zheng Y.S.,2017,Optimization of ISSR-PCR system of Dendrocala musminor var.amoenus based on orthogonal design method,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2278-2285(瞿印权,徐雯,沈露,陈剑成,何天友,魏建文,荣俊冬,郑郁善,2017,花吊丝竹ISSR-PCR反应体系的正交优化,分子植物育种,15(6):2278-2285)
Ren F.M.,Hu K.Z.,Liu Y.Q.,Jiao Y.X.,Liu J.,Luo M.,and Quan J.,2014,Optimization for ISSR-PCR system of traditional Chinese medicine Lysimachia christinae by orthogonal design,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),39(12):2233-2238(任风鸣,胡开治,刘燕琴,焦雁翔,刘杰,罗敏,全健,2014,传统中药金钱草ISSR-PCR反应体系的正交优化研究,中国中药杂志,39(12):2233-2238)
Su X.J.,Li J.X.,Xu H.J.,Qu Y.Y.,and Chen Q.J.,2015,Optimization of ISSR-PCR system of lavender based on orthogo nal design method,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):641-645(苏秀娟,刘建喜,徐海江,曲延英,陈全家,2015,薰衣草ISSR-PCR反应体系的正交优化,分子植物育种,13(3):641-645)
Xu W.,Qu Y.Q.,Han X.,He T.Y.,Rong J.D.,and Zheng Y.S.,2017,Optimization of ISSR-PCR reaction system on Dendrocalamus oldhami by orthogonal design,and selection of primer,shengw jishu tongbao(Biotechnology Bulletin),33(8):103-110(徐雯,瞿印权,韩笑,何天友,荣俊冬,郑郁善,2017,正交设计优化绿竹ISSR-PCR体系和引物筛选,生物技术通报,33(8):103-110)
Zhou P.,Guo S.L.,and Yin L.P.,2013,Status of research on taxonomy and systematics of the genus Echinochloa,Zacao Kexue(Weed Science),31(1):1-4(周平,郭水良,印丽萍,2013,稗属植物的分类与系统学研究概况,杂草科学,31(1):1-4)
Zhang Z.C.,Gu T.,Yang X.,Peng Q.,Bo L.Y.,and Li Y.F.,2017,Effects of barnyardgrass with different resistance on grain yield of rice and their physiological characteristics,Henongxue Bao(Journal of Nuclear Agricultural Sciences),31(8):1594-1603(张自常,谷涛,杨霞,彭琼,柏连阳,李永丰,2017,不同抗性稗草对水稻产量及其生理特性的影响,核农学报,31(8):1594-1603)
Zhang J.L.,Wu S.,Shi X.G.,Li B.T.,and Tang L.M.,2015,Influence of barnyardgrass(Echinochloa crusgalli)on the growth of double-cropping paddy rice and its economic threshold,Caoye Xuebao(Acta Prataculturae Sinica),24(8):44-52(张纪利,吴尚,石绪根,李保同,汤丽梅,2015,稗草对双季稻生长的影响及其防除经济阈值研究,草业学报,24(8):44-52)
Zuo C.P.,Ji M.S.,Zang X.X.,Chen S.H.,and Du Y.,2017,The resistance level and acetyl co-enzyme A carboxylase activity of Echinochloa phyllopogon populations to metamifop in paddies,Zhiwu Baohu Xuebao(Journal of Plant Protection),44(6):1040-1045(左平春,纪明山,臧晓霞,陈仕红,杜颖,2017,稻田稗草对噁唑酰草胺的抗药性水平和ACCase活性,植物保护学报,44(6):1040-1045)
Zhang W.R.,Wu S.J.,and Wang X.P.,2018,Research advances in germplasm resources and applications of Echinochloa,Pratacultural Science,35(1):165-175(张婉茹,吴姝菊,王晓萍,2018,稗属植物种质资源及应用价值研究进展,草业科学,35(1):165-175)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genomic fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183
Huang X.Y.,Tang H.,Kong D.X.,Wang M.L.,Shi Y.C.,and Zou R.,2013,Establishment and optimization of ISSR-PCRreaction conditions in Rubus suavissimus S.Lee,Jiyinzuzue Yu Yingyong Shengwuxue(Genomics and Applied Biology),32(4):526-532(黄夕洋,唐辉,孔德鑫,王满莲,史艳财,邹蓉,2013,广西甜茶ISSR-PCR反应条件的建立与优化,基因组学与应用生物学,32(4):526-532)
He Z.W.,Liu Y.S.,Chen L.H.,Cao M.H.,and Xia J.H.,1998,Orthogonal design-direct analysis for PCR optimization,Hunan Yike Daxue Xuebao(Bulletin of Hunan Medical University),23(4):403-404(何正文,刘运生,陈立华,曹美鸿,夏家辉,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)
Hu Y.P.,Wang L.,Bao R.,Shi L.,Xu R.H.,and Li Y.,2014,Optimizing ISSR-PCR system of Carex atrofusca using orthogonal design,Caodi Xuebao(Acta Agrestia Sinica),22(4):911-914,918(胡延萍,王莉,包蕊,石琳,旭荣花,李毅,2014,正交设计优化黑褐苔草ISSR-PCR反应体系,草地学报,22(4):911-914,918)
Liu D.H.,Lu Y.L.,Lou Y.X.,and Guo S.L.,2016,Optimization of ISSR-PCR reaction system of Echinochloa by orthogonal design,Shanghai Nongye Xuebao(Acta Agriculturae Shanghai),32(4):17-21(刘德好,陆永良,娄玉霞,郭水良,2016,正交试验法优化稗属植物ISSR-PCR反应体系,上海农业学报,32(4):17-21)
Li R.H.,Xia Y.S.,Liu S.Z.,Sun L.L.,Guo P.G.,Miao S.Y.,and Chen J.H.,2009,CTAB-improved method of DNA extractionin plant,Shiyanshi Yanjiu Yu Tansuo(Research and Explorationin Laboratory),28(9):14-16(李荣华,夏岩石,刘顺枝,孙莉丽,郭培国,缪绅裕,陈健辉,2009,改进的CTAB提取植物DNA方法,实验室研究与探索,28(9):14-16)
Jing Q.Y.,Jiao Z.Z.,Liu L.K.,Shi H.,Feng L.,Li W.,and Liu Y.G.,2014,The resistance of barnyardgrass and amaranthus retroflexus to four common used herbicides in corn fields in Heilongjiang province,Zuowu Xuebao(Crops),(5):128-132(井秋月,焦梓洲,刘兰坤,施慧,冯蕾,李威,刘亚光,2014,黑龙江省玉米田稗草与反枝苋对四种常用除草剂的抗药性测定,作物杂志,(5):128-132)
Qu Y.Q.,Xu W.,Shen L.,Chen J.C.,He T.Y.,Wei J.W.,Rong J.D.,and Zheng Y.S.,2017,Optimization of ISSR-PCR system of Dendrocala musminor var.amoenus based on orthogonal design method,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2278-2285(瞿印权,徐雯,沈露,陈剑成,何天友,魏建文,荣俊冬,郑郁善,2017,花吊丝竹ISSR-PCR反应体系的正交优化,分子植物育种,15(6):2278-2285)
Ren F.M.,Hu K.Z.,Liu Y.Q.,Jiao Y.X.,Liu J.,Luo M.,and Quan J.,2014,Optimization for ISSR-PCR system of traditional Chinese medicine Lysimachia christinae by orthogonal design,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),39(12):2233-2238(任风鸣,胡开治,刘燕琴,焦雁翔,刘杰,罗敏,全健,2014,传统中药金钱草ISSR-PCR反应体系的正交优化研究,中国中药杂志,39(12):2233-2238)
Su X.J.,Li J.X.,Xu H.J.,Qu Y.Y.,and Chen Q.J.,2015,Optimization of ISSR-PCR system of lavender based on orthogo nal design method,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(3):641-645(苏秀娟,刘建喜,徐海江,曲延英,陈全家,2015,薰衣草ISSR-PCR反应体系的正交优化,分子植物育种,13(3):641-645)
Xu W.,Qu Y.Q.,Han X.,He T.Y.,Rong J.D.,and Zheng Y.S.,2017,Optimization of ISSR-PCR reaction system on Dendrocalamus oldhami by orthogonal design,and selection of primer,shengw jishu tongbao(Biotechnology Bulletin),33(8):103-110(徐雯,瞿印权,韩笑,何天友,荣俊冬,郑郁善,2017,正交设计优化绿竹ISSR-PCR体系和引物筛选,生物技术通报,33(8):103-110)
Zhou P.,Guo S.L.,and Yin L.P.,2013,Status of research on taxonomy and systematics of the genus Echinochloa,Zacao Kexue(Weed Science),31(1):1-4(周平,郭水良,印丽萍,2013,稗属植物的分类与系统学研究概况,杂草科学,31(1):1-4)
Zhang Z.C.,Gu T.,Yang X.,Peng Q.,Bo L.Y.,and Li Y.F.,2017,Effects of barnyardgrass with different resistance on grain yield of rice and their physiological characteristics,Henongxue Bao(Journal of Nuclear Agricultural Sciences),31(8):1594-1603(张自常,谷涛,杨霞,彭琼,柏连阳,李永丰,2017,不同抗性稗草对水稻产量及其生理特性的影响,核农学报,31(8):1594-1603)
Zhang J.L.,Wu S.,Shi X.G.,Li B.T.,and Tang L.M.,2015,Influence of barnyardgrass(Echinochloa crusgalli)on the growth of double-cropping paddy rice and its economic threshold,Caoye Xuebao(Acta Prataculturae Sinica),24(8):44-52(张纪利,吴尚,石绪根,李保同,汤丽梅,2015,稗草对双季稻生长的影响及其防除经济阈值研究,草业学报,24(8):44-52)
Zuo C.P.,Ji M.S.,Zang X.X.,Chen S.H.,and Du Y.,2017,The resistance level and acetyl co-enzyme A carboxylase activity of Echinochloa phyllopogon populations to metamifop in paddies,Zhiwu Baohu Xuebao(Journal of Plant Protection),44(6):1040-1045(左平春,纪明山,臧晓霞,陈仕红,杜颖,2017,稻田稗草对噁唑酰草胺的抗药性水平和ACCase活性,植物保护学报,44(6):1040-1045)
Zhang W.R.,Wu S.J.,and Wang X.P.,2018,Research advances in germplasm resources and applications of Echinochloa,Pratacultural Science,35(1):165-175(张婉茹,吴姝菊,王晓萍,2018,稗属植物种质资源及应用价值研究进展,草业科学,35(1):165-175)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genomic fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183