芦笋ISSR反应体系的建立及优化
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摘要
通过正交试验建立芦笋ISSR优化反应体系,并对其反应程序进行优化。实验结果表明,优化的扩增体系:25μL的反应体系中含150 ng的模板DNA、0.3μmol/L引物、15μL 2×Mastermix;优化的反应程序:94℃预变性5 min;94℃变性45 s,设定温度退火60 s,72℃延伸45 s,循环30次;然后72℃延伸10 min,4℃保存;筛选到了21条引物并确定其最优退火温度。实验结果为芦笋资源遗传多样性评价的研究奠定基础。
The optimum ISSR reaction system in A.officinalis was established by orthogonal test,then its reaction process was optimized.The result showed that the optimized amplification system was 150 ng temple DNA in the total volume of 25 μL ISSR system,0.3 μmol/L primer,and 15 μL 2× Mastermix.The optimized PCR procedure was: pre-denaturation at 94℃ for 5 min,denaturation at 94℃ for 45 s,setting temperature annealing for 60 s,extension at 72℃ for 45 s,and repeated for 30 cycles; then extension at 72℃ for 10 min,and remaining at 4℃.Based on this optimized system,21 primers were screened and the optimal annealing temperature was set.The results could lay the foundation for the genetic diversity evaluation of A.officinalis.
The optimum ISSR reaction system in A.officinalis was established by orthogonal test,then its reaction process was optimized.The result showed that the optimized amplification system was 150 ng temple DNA in the total volume of 25 μL ISSR system,0.3 μmol/L primer,and 15 μL 2× Mastermix.The optimized PCR procedure was: pre-denaturation at 94℃ for 5 min,denaturation at 94℃ for 45 s,setting temperature annealing for 60 s,extension at 72℃ for 45 s,and repeated for 30 cycles; then extension at 72℃ for 10 min,and remaining at 4℃.Based on this optimized system,21 primers were screened and the optimal annealing temperature was set.The results could lay the foundation for the genetic diversity evaluation of A.officinalis.
引文
Chen G.Y.,ed.,2010,Asparagus research and industrial development in China,Chinese Agricultural Press,Beijing,China,pp.3-9(陈光宇,编著,2010,中国芦笋研究与产业发展,中国农业出版社,中国,北京,pp.3-9)
Chen H.L.,Gao J.M.,Zhang S.Q.,Xi J.G.,Zheng J.L.,Liu Q.L.,and Yi K.X.,2015,Establishment and optimization of ISSRreaction system in sisal,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),(2):20-23(陈河龙,高建明,张世清,习金根,郑金龙,刘巧莲,易克贤,2015,剑麻ISSR反应体系的建立及其优化,贵州农业科学,(2):20-23)
Li F.F.,Yang H.J.,and Cui D.F.,2014,Genetic relationships among11 species of medicago and relative species using microsatellite(SSR and ISSR)markers,Zhongshan Daxue Xuebao(Acta Scientiarum Naturalium Universitatis Sunyatseni),53(1):113-120(李飞飞,羊海军,崔大方,2014,利用SSR及ISSR分子标记研究苜蓿属及其近缘植物的亲缘关系,中山大学学报(自然科学版),53(1):113-120)
Li G.S.,Cao F.X.,Peng J.Q.,Xu W.,Hu S.X.,and Peng Y.C.,2014,Establishment and optimization of ISSR-PCR reaction system of Camellia meiocarpa hu,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry&Technology),34(4):36-42(李国帅,曹福祥,彭继庆,胥文,胡双喜,彭滟钞,2014,野生小果油茶ISSR-PCR反应体系的建立与优化,中南林业科技大学学报,34(4):36-42)
Liu S.Q.,Wu F.Z.,and Liu S.W.,2012,Inter-simple sequence repeat and its application in horticultural crop research,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),43(4):145-150(刘淑芹,吴凤芝,刘守伟,2012,园艺作物的ISSR分子标记研究及应用,东北农业大学学报,43(4):145-150)
Ou L.J.,Yan W.,Liao Y.X.,Liang Y.H.,and She C.W.,2011,Establishment and optimization of ISSR-PCR reaction system in Asparagus cochinchinensis,Zhongcaoyao(Chinese Traditional and Herbal Drugs),42(2):353-357(欧立军,颜旺,廖亚西,梁云浩,佘朝文,2011,天门冬ISSR分子标记技术的建立与体系优化,中草药,42(2):353-357)
Sheng W.T.,Zhou J.S.,Tang Y.P.,Luo S.C.,Zhu Y.L.,and Chen G.Y.,2010,Optimization of extraction of total dna and ISSR-PCR system in asparagus officinalis,Jiangxi Nongye Xuebao(Acta Agriculturae Jiangxi),22(9):38-41(盛文涛,周劲松,汤泳萍,罗绍春,朱友林,陈光宇,2010,芦笋基因组DNA提取与ISSR反应体系的优化,江西农业学报,22(9):38-41)
Zhu Y.F.,Zhu S.J.,Li Y.P.,Ma W.G.,Zheng Y.Y.,and Hu J.,2010,Application of issr molecular marker in plant germplasm research,Zhongzi(Seed),29(2):55-59(朱岩芳,祝水金,李永平,马文广,郑昀晔,胡晋,2010,ISSR分子标记技术在植物种质资源研究中的应用,种子,29(2):55-59)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183
Chen H.L.,Gao J.M.,Zhang S.Q.,Xi J.G.,Zheng J.L.,Liu Q.L.,and Yi K.X.,2015,Establishment and optimization of ISSRreaction system in sisal,Guizhou Nongye Kexue(Guizhou Agricultural Sciences),(2):20-23(陈河龙,高建明,张世清,习金根,郑金龙,刘巧莲,易克贤,2015,剑麻ISSR反应体系的建立及其优化,贵州农业科学,(2):20-23)
Li F.F.,Yang H.J.,and Cui D.F.,2014,Genetic relationships among11 species of medicago and relative species using microsatellite(SSR and ISSR)markers,Zhongshan Daxue Xuebao(Acta Scientiarum Naturalium Universitatis Sunyatseni),53(1):113-120(李飞飞,羊海军,崔大方,2014,利用SSR及ISSR分子标记研究苜蓿属及其近缘植物的亲缘关系,中山大学学报(自然科学版),53(1):113-120)
Li G.S.,Cao F.X.,Peng J.Q.,Xu W.,Hu S.X.,and Peng Y.C.,2014,Establishment and optimization of ISSR-PCR reaction system of Camellia meiocarpa hu,Zhongnan Linye Keji Daxue Xuebao(Journal of Central South University of Forestry&Technology),34(4):36-42(李国帅,曹福祥,彭继庆,胥文,胡双喜,彭滟钞,2014,野生小果油茶ISSR-PCR反应体系的建立与优化,中南林业科技大学学报,34(4):36-42)
Liu S.Q.,Wu F.Z.,and Liu S.W.,2012,Inter-simple sequence repeat and its application in horticultural crop research,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),43(4):145-150(刘淑芹,吴凤芝,刘守伟,2012,园艺作物的ISSR分子标记研究及应用,东北农业大学学报,43(4):145-150)
Ou L.J.,Yan W.,Liao Y.X.,Liang Y.H.,and She C.W.,2011,Establishment and optimization of ISSR-PCR reaction system in Asparagus cochinchinensis,Zhongcaoyao(Chinese Traditional and Herbal Drugs),42(2):353-357(欧立军,颜旺,廖亚西,梁云浩,佘朝文,2011,天门冬ISSR分子标记技术的建立与体系优化,中草药,42(2):353-357)
Sheng W.T.,Zhou J.S.,Tang Y.P.,Luo S.C.,Zhu Y.L.,and Chen G.Y.,2010,Optimization of extraction of total dna and ISSR-PCR system in asparagus officinalis,Jiangxi Nongye Xuebao(Acta Agriculturae Jiangxi),22(9):38-41(盛文涛,周劲松,汤泳萍,罗绍春,朱友林,陈光宇,2010,芦笋基因组DNA提取与ISSR反应体系的优化,江西农业学报,22(9):38-41)
Zhu Y.F.,Zhu S.J.,Li Y.P.,Ma W.G.,Zheng Y.Y.,and Hu J.,2010,Application of issr molecular marker in plant germplasm research,Zhongzi(Seed),29(2):55-59(朱岩芳,祝水金,李永平,马文广,郑昀晔,胡晋,2010,ISSR分子标记技术在植物种质资源研究中的应用,种子,29(2):55-59)
Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183