小麦种子规模化DNA提取及检测体系的优化
|
摘要
以小麦种子分子检测农业行业标准(NY/T 2859-2015)为基础,开发小麦种子快速、规模化提取DNA方法,优化反应体系中各组分含量,为小麦真实性快速执法提供技术支撑。通过对SDS、CTAB、高盐低p H、快速提取和试剂盒5种方法提取的DNA质量、浓度和PCR扩增效果进行比较,发现改进的高盐低p H方法提取种子的DNA质量、浓度能够满足小麦真实性鉴定中42对SSR引物重复鉴定的需求,是利用96孔深孔板和自动化移液工作站规模化、高通量提取DNA的较优方法。进一步优化结果显示该方法在65℃温浴条件下比室温的浓度高出25%,但两种温度条件下提取的DNA质量及浓度均能够满足品种鉴定的需求;沉淀时用0.5倍提取液体积的预冷异丙醇沉淀浓度最高,用室温的异丙醇沉淀的最佳体积是提取液体积的0.6倍。对标准中42对引物的最佳引物浓度和模板浓度均进行了优化,综合所有42对引物的优化结果发现,反应体系为20μL时,引物终浓度为0.437 5μmol/L,模板终浓度为10 ng/μL时扩增效率相对较高,扩增产物稳定,能够满足多重电泳的需求。
Based on the industry standard of wheat authenticity SSR( NY/T 2859-2015),this study developed a rapid and large-scale DNA extraction method for wheat seeds,and optimized the content of components in the reaction system toprovide technical support for rapid enforcement of wheat authenticity law. By comparing the DNA quality,concentration and PCR amplification effect that extracted by five methods such as SDS,CTAB,high-salt low-pH,rapid extraction and kit,it was found that the improved method of the high-salt low-pH could meet the demand of 42 SSR primers for repeated identification in wheat authenticity identification. It was the better method for 96-well deephole plate and automated liquid transfer station. The results of further optimization showed that the concentration of this method at room temperature was 25% higher than that at at 65℃,but the quality and concentration of DNA extracted at both temperatures could meet the requirements of variety identification. The highest concentration of DNA was obtained with 0. 5-fold volume of precooled isopropanol,and with 0. 6-fold volume of isopropanol at room temperature. The optimal primer concentration and template concentration of 42 pairs of primers in the standard were optimized. The results showed that when the reaction system was 20 μL,the final primer concentration was 0.437 5μmol/L,and the final template concentration was 10 ng/μL,the amplification efficiency was relatively high,and the amplification products were stable,which could meet the needs of multiple electrophoresis.
Based on the industry standard of wheat authenticity SSR( NY/T 2859-2015),this study developed a rapid and large-scale DNA extraction method for wheat seeds,and optimized the content of components in the reaction system toprovide technical support for rapid enforcement of wheat authenticity law. By comparing the DNA quality,concentration and PCR amplification effect that extracted by five methods such as SDS,CTAB,high-salt low-pH,rapid extraction and kit,it was found that the improved method of the high-salt low-pH could meet the demand of 42 SSR primers for repeated identification in wheat authenticity identification. It was the better method for 96-well deephole plate and automated liquid transfer station. The results of further optimization showed that the concentration of this method at room temperature was 25% higher than that at at 65℃,but the quality and concentration of DNA extracted at both temperatures could meet the requirements of variety identification. The highest concentration of DNA was obtained with 0. 5-fold volume of precooled isopropanol,and with 0. 6-fold volume of isopropanol at room temperature. The optimal primer concentration and template concentration of 42 pairs of primers in the standard were optimized. The results showed that when the reaction system was 20 μL,the final primer concentration was 0.437 5μmol/L,and the final template concentration was 10 ng/μL,the amplification efficiency was relatively high,and the amplification products were stable,which could meet the needs of multiple electrophoresis.
引文
[1]支巨振,毕辛华,杜克敏,等.GB/T 3543.5-1995农作物种子检验规程真实性和品种纯度鉴定[S].北京:中国标准出版社,1995.Zhi J Z,Bi X H,Du K M,et al..GB/T 3543.5-1995 Rules of agricultural seed testing-verification of genuineness and cultivar[S].Beijing:Standards Press of China,1995.
[2]赵昌平,支巨振,邱军,等.NY/T 2859-2015,主要农作物品种真实性SSR分子标记检测普通小麦[S].北京:中国标准出版社,2015.Zhao C P,Zhi J Z,Qiu J,et al..NY/T 2859-2015 Variety genuineness testing of main crops with SSR markers---Wheat(Triticum aestivum L.)[S].Beijing:Standards Press of China,2015.
[3]杨少辉,张丽娟,段会军,等.大豆种子DNA的提取方法[J].大豆科学,2003,22(2).Yang S H,Zhang L J,Duan H J,et al..Rapid extraction of soybean DNA for PCR from dry seed[J].Soybean Sci.,2003,22(2).
[4]王芳.大麦干种子DNA提取研究[J].安徽农业科学,2007,35(26):8132-8133.Wang F.Study on DNA extraction in dry barley seed[J].J.Anhui Agric.Sci.,2007,35(26):8132-8133.
[5]郭景伦,赵久然,王凤格.适用于SSR分子标记的玉米单粒种子DNA快速提取新方法[J].玉米科学,2005,13(2):16-17.Guo J L,Zhao J R,Wang F G.A new rapid DNA extraction method from maize single dry seed suitable for SSR analysis[J].J.Maize Sci.,2005,13(2):16-17.
[6]郎需勇,刘伟霞,杨亮,等.一种快速提取棉花干种子基因组DNA的新方法[J].棉花学报,2014,26(1):87-94.Lang X Y,Liu W X,Yang L,et al..A new rapid DNAextraction method suitable for dry cotton seed[J].Cotton Sci.,2014,26(1):87-94.
[7]杨俊宝,彭正松,赵梅,等.一种从小麦干种子中快速提取DNA的优化方法[J].西华师范大学学报(自然科学版),2006,27(1):48-52.Yang J B,Peng Z S,Zhao M.An optimum and rapid DNAextraction method from dry seeds of wheat[J].J.China West Norm.Univ.(Nat.Sci.),2006,27(1):48-52.
[8]李荣华,夏岩石,刘顺枝,等.改进的CTAB提取植物DNA方法[J].实验室研究与探索,2009,28(9):14-16.Li R H,Xia Y S,Liu S Z,et al..CTAB-improved method of DNA extraction in plant[J].Res.Exploration Laboratory,2009,28(9):14-16.
[9]梁雪莲,郑奕雄,陈晓玲,等.花生DNA提取方法比较[J].生物技术,2007,17(1):41-44.Liang X L,Zheng Y X,Chen X L,et al..Comparison of different methods of DNA extraction of peanut leave[J].Biotechnology,2007,17(1):41-44.
[10]许理文,王凤格,赵久然,等.高盐低p H值法提取玉米基因组DNA的研究[J].玉米科学,2009,17(1):59-61.Xu L W,Wang F G,Zhao J R,et al..Studies on genomic DNA extraction of maize with high salt,low p H method[J].J.Maize Sci.,2009,17(1):59-61.
[11]师丽红,杨文香,刘大群.小麦基因组的一种简易提取方法[J].中国农学通报,2010,26(19):22-26.Shi L H,Yang W X,Liu D Q.One simple method for extracting the wheat genome[J].Chin.Agric.Sci.Bull.,2010,26(19):22-26.
[12]易红梅,王凤格,赵久然,等.玉米品种SSR标记毛细管电泳荧光检测法与变性PAGE银染检测法的比较研究[J].华北农学报,2006,21(5):64-67.Yi H M,Wang F G,Zhao J R,et al..Comparison of two maize SSR detection methods:capillary electrophoresis with fluorescence detection method and denaturing PAGE silverstaining detection method[J].Acta Agric.Boreal.Sin.,2006,21(5):64-67.
[13]王凤格,李欣,杨扬,等.植物品种SSR指纹分析专用软件SSR Analyser的研发[J].中国农业科学,2018,51(12):2248-2262.Wang F G,Li X,Yang Y,et al..SSR Analyser:A special software suitable for SSR fingerprinting of plant varieties[J].Sci.Agric.Sin.,2018,51(12):2248-2262.
[14]张富丽,陶李,佟洪金,等.从干种子中快速获取高质量DNA的高盐提取方法[J].中国农学通报,2011,27(24):98-102.Zhang F L,Tao L,Tong H J,et al..A method of simple and rapid salt-extraction of high quality genomic DNA from plant seeds[J].Chin.Agric.Sci.Bull.,2011,27(24):98-102.
[2]赵昌平,支巨振,邱军,等.NY/T 2859-2015,主要农作物品种真实性SSR分子标记检测普通小麦[S].北京:中国标准出版社,2015.Zhao C P,Zhi J Z,Qiu J,et al..NY/T 2859-2015 Variety genuineness testing of main crops with SSR markers---Wheat(Triticum aestivum L.)[S].Beijing:Standards Press of China,2015.
[3]杨少辉,张丽娟,段会军,等.大豆种子DNA的提取方法[J].大豆科学,2003,22(2).Yang S H,Zhang L J,Duan H J,et al..Rapid extraction of soybean DNA for PCR from dry seed[J].Soybean Sci.,2003,22(2).
[4]王芳.大麦干种子DNA提取研究[J].安徽农业科学,2007,35(26):8132-8133.Wang F.Study on DNA extraction in dry barley seed[J].J.Anhui Agric.Sci.,2007,35(26):8132-8133.
[5]郭景伦,赵久然,王凤格.适用于SSR分子标记的玉米单粒种子DNA快速提取新方法[J].玉米科学,2005,13(2):16-17.Guo J L,Zhao J R,Wang F G.A new rapid DNA extraction method from maize single dry seed suitable for SSR analysis[J].J.Maize Sci.,2005,13(2):16-17.
[6]郎需勇,刘伟霞,杨亮,等.一种快速提取棉花干种子基因组DNA的新方法[J].棉花学报,2014,26(1):87-94.Lang X Y,Liu W X,Yang L,et al..A new rapid DNAextraction method suitable for dry cotton seed[J].Cotton Sci.,2014,26(1):87-94.
[7]杨俊宝,彭正松,赵梅,等.一种从小麦干种子中快速提取DNA的优化方法[J].西华师范大学学报(自然科学版),2006,27(1):48-52.Yang J B,Peng Z S,Zhao M.An optimum and rapid DNAextraction method from dry seeds of wheat[J].J.China West Norm.Univ.(Nat.Sci.),2006,27(1):48-52.
[8]李荣华,夏岩石,刘顺枝,等.改进的CTAB提取植物DNA方法[J].实验室研究与探索,2009,28(9):14-16.Li R H,Xia Y S,Liu S Z,et al..CTAB-improved method of DNA extraction in plant[J].Res.Exploration Laboratory,2009,28(9):14-16.
[9]梁雪莲,郑奕雄,陈晓玲,等.花生DNA提取方法比较[J].生物技术,2007,17(1):41-44.Liang X L,Zheng Y X,Chen X L,et al..Comparison of different methods of DNA extraction of peanut leave[J].Biotechnology,2007,17(1):41-44.
[10]许理文,王凤格,赵久然,等.高盐低p H值法提取玉米基因组DNA的研究[J].玉米科学,2009,17(1):59-61.Xu L W,Wang F G,Zhao J R,et al..Studies on genomic DNA extraction of maize with high salt,low p H method[J].J.Maize Sci.,2009,17(1):59-61.
[11]师丽红,杨文香,刘大群.小麦基因组的一种简易提取方法[J].中国农学通报,2010,26(19):22-26.Shi L H,Yang W X,Liu D Q.One simple method for extracting the wheat genome[J].Chin.Agric.Sci.Bull.,2010,26(19):22-26.
[12]易红梅,王凤格,赵久然,等.玉米品种SSR标记毛细管电泳荧光检测法与变性PAGE银染检测法的比较研究[J].华北农学报,2006,21(5):64-67.Yi H M,Wang F G,Zhao J R,et al..Comparison of two maize SSR detection methods:capillary electrophoresis with fluorescence detection method and denaturing PAGE silverstaining detection method[J].Acta Agric.Boreal.Sin.,2006,21(5):64-67.
[13]王凤格,李欣,杨扬,等.植物品种SSR指纹分析专用软件SSR Analyser的研发[J].中国农业科学,2018,51(12):2248-2262.Wang F G,Li X,Yang Y,et al..SSR Analyser:A special software suitable for SSR fingerprinting of plant varieties[J].Sci.Agric.Sin.,2018,51(12):2248-2262.
[14]张富丽,陶李,佟洪金,等.从干种子中快速获取高质量DNA的高盐提取方法[J].中国农学通报,2011,27(24):98-102.Zhang F L,Tao L,Tong H J,et al..A method of simple and rapid salt-extraction of high quality genomic DNA from plant seeds[J].Chin.Agric.Sci.Bull.,2011,27(24):98-102.