牡丹基因组AFLP银染反应体系的建立和优化

采用改进的SDS方法,从牡丹成熟叶片中提取基因组DNA,所得DNA样品的A260/A280值在1.7～1.8之间,琼脂糖凝胶电泳主带清晰、DNA纯度高、较少降解、不含酶反应抑制剂,可被MseⅠ和PstⅠ两种限制性内切酶完全酶切.通过酶切连接、预扩增、选择性扩增、银染等试验过程,成功建立了牡丹品种AFLP银染反应体系,得到了清晰的指纹图谱.对扩增结果进行聚类分析,得到了23个牡丹品种的聚类分析图.     

梅花基因组AFLP银染反应体系的建立和优化

采用改进的SDS DNA提取方法从梅花的嫩叶和老叶中提取获得总DNA .所得DNA样品的OD2 6 0 OD2 80 值在1 7～ 1 9之间 ,琼脂糖凝胶上主带清晰、较少降解、样品纯度高、蛋白去除干净且得率高 .该体系对原提取程序作了多处简化 ,使操作简便、快捷、高效 ,适合于梅花DNA提取 ,所提取的DNA样品不含PCR反应抑制剂及其他酶反应抑制剂 ,可被限制性内切酶MseⅠ和EcoRⅠ完全酶切 ,适合AFLP PCR反应应用 ,得到清晰的指纹式样 .     

蜡梅AFLP反应体系优化及引物筛选

通过对影响AFLP反应体系主要因素的优化,建立了蜡梅的AFLP反应体系,并筛选出了适宜该体系分析的引物.结果表明,20μL蜡梅AFLP最佳酶切体系为模板600 ng DNA,3 U Pst I和3 U Mse I,在37℃下双酶切2 h;在20μL最佳连接体系中酶切产物为15μL,3 U T4连接酶,0.25μmol.L-1 Pst I接头,2.5μmol.L-1 Mse I接头,1μL 10×T4 Buffer,在22℃下连接10 h;在20μL最佳预扩反应体系中稀释10倍的连接产5μL,2.0mmol.L-1 Mg2+,2 U Taq酶,300μmol.L-1dNTP,0.5μmol.L-1 Pst I和Mse I引物(P+AGA/M+ATC);在20μL最佳选择性扩增反应体系中5μL稀释20倍的预扩增产物,2.0 mmol.L-1的Mg2+,2 U Taq酶,300μmol.L-1 dNTP,0.5μmol.L-1 Pst I和Mse I引物(P+AGA/M+ATC).最后,利用上面的体系筛选出了96对适宜于蜡梅AFLP分析的引物.     

泡桐AFLP反应体系的建立及引物筛选

以豫杂一号泡桐为材料,通过对影响AFLP技术体系的各主要因素的研究,建立了适于泡桐AFLP分析的技术体系.结果表明最佳酶切体系(20μL)为500 ng模板DNA,3 U的Pst 1和Mse I,在37℃下双酶切3 h;20μL最佳连接体系中为酶切产物15 μL,0.25 μmol·L-1Pst I接头,2.5 μmol·L-1 Mse I接头,1 μL 10×T4 Buff-er,2 U T4连接酶,22℃连接18 h;20 μL最佳预扩反应体系中5 μL稀释10倍的连接产物,100 μmol·L-1dNTP,2 U Taq酶,250 μmol.L-1 Pst I和Mse I引物(P+AGT/M+AGT),2 μL 10×PCR Buffer.20 μL最佳选择性扩增反应体系中5 μL稀释20倍预扩增产物,100 μmol·L-1dNTP,2 U Taq酶,350 μmol·L-1 Pst I和Mse I引物(P+AGT/M+AGT),2 μL 10×PCR Buffer.最后,筛选出了97对适宜于泡桐AFLP分析的引物.     

割手密AFLP分子标记技术体系建立与优化

【目的】建立和优化割手密AFLP分子标记技术体系,为割手密遗传多样性分析、遗传图谱构建提供技术支持。【方法】以广西割手密GXS87—16、GXS85．30、GXS79—9、GXS96、GXS112、GXS212为材料,利用改良SDS法提取DNA,并用EcoR I和Mse I酶切,连接接头后,采用正交试验设计对影响预扩增反应和选择性扩增反应的主要成分如Mg^2＋、模板DNA、引物、dNTP、Taq聚合酶浓度进行优化。【结果】样品DNA用限制性内切酶＆0RI和MseI各3u于PCR仪过夜可完全酶切。经正交设计优化,较佳的预扩增体系包含0．4μL dNTPs（20mmol／mL）,1．6μLMg^2＋（25mmol／mL）,2．0μL EcoRI—P（5pmol／mL）,2．0IxLMseI-P（5pmol／mL）,1UTaq酶（1U／μL）,DNA模板稀释10倍;选择性扩增体系包含0．4μL dNTPS（20mmol／mL）,0．8μLMg^2＋（25mmol／mL）,1．0μL EcoR I—AAG（6pmol／mL）,1．0μL Mse I-CAG（6pmol／mL）,3U Taq酶（1U／μL）,DNA模板稀释20倍。以对优化反应体系扩增获得的PcR产物用5％变性聚丙烯酰胺凝胶电泳和银染后,可获得清晰的多态性指纹图谱。【结论】建立的割手密AFLP分子标记技术体系具有扩增条带清晰、多态性丰富的特点,可为构建割手密高密度遗传图谱提供技术支持。     

悬铃木AFLP分子标记技术体系的建立

采用改进的CTAB方法,从悬铃木叶片中提取基因组DNA,所得DNA样品的A260/A280值为1.7～1.9,琼脂糖凝胶电泳主带清晰、DNA纯度高、较少降解、不含酶反应抑制剂,可被MseⅠ和PstⅠ两种限制性内切酶完全酶切,从20对引物中筛选出6对带型分布均匀、多态性高且分辨能力强的引物,分别为: M74P50,M89P57,M74P26,M36P16,M80P66,M23P66.通过酶切连接、预扩增、选择性扩增、银染等试验过程,成功建立了悬铃木AP反应体系,得到了清晰的指纹图谱,试验结果重复性好.     

凉粉草基因组DNA提取与分析

[目的]为分子生物学研究提供高质量、高产量的凉粉草总DNA。[方法]采用改良CTAB法提取总DNA,通过测定OD260/OD280值、琼脂糖凝胶电泳和RAPD扩增对所得DNA的浓度和质量进行检测,考察该方法对DNA的提取效果。[结果]改良CTAB法提取的总DNAOD260/OD280值为1.6～2.0;电泳条带清晰,完整性好,纯度高,总DNA分子量与λDNA相近;以所提DNA为模板进行RAPD扩增时,可得到稳定的扩增条带。[结论]改良CTAB法适合凉粉草基因组DNA的提取。     

华北落叶松AFLP反应体系的建立和优化

[目的]建立和优化华北落叶松（Larixprincipis-rupprechtii Mayr.）的AFLP反应体系。[方法]以华北落叶松为材料,对其AFLP分析过程中的5个主要影响因素（酶切时间、内切酶用量、DNA模板用量、连接产物和预扩增产物稀释倍数）进行了研究。[结果]确立了适于华北落叶松AFLP分析的最佳反应体系,即在反应体积均为20.0μl的前提下,酶切时间为7 h（37℃3.5 h;65℃3.5 h）,EcoRⅠ和MseⅠ内切酶用量均为1 U,DNA模板用量为3.0μl（50 ng/μl）,连接产物稀释10倍取5.0μl用于预扩增,预扩增产物稀释70倍取4.0μl用于选择性扩增。[结论]采用该体系得到的电泳条带清晰可辨、多态性好、重复性强,可用于对华北落叶松遗传多样性和分子进化特征的深入研究。     

枸杞属AFLP分析体系优化与建立

以枸杞属种质资源为试材,对AFLP分析过程中包括DNA的提取质量和浓度、Mse I/EcoR I以及T4 DNA连接酶的浓度、反应时间等影响因素进行了研究.建立了一种适于枸杞AFLP银染技术的优化体系.该体系中各优化因素为:模板DNA的用量为300～400 ng,酶切体系中Mse I和EcoR I各加入3 U,T4 DNA连接酶加入2.0U,采用酶切与连接在同一体系中一步完成,其酶切温度为37℃,反应时间为5 h.与22℃连接过夜.并对预扩增、选择性扩增和银染的效果进行了检测,以引物M+CAC/E+AGG构建了枸杞种质资源的AFLP指纹图谱,该图谱扩增带多,多态性强,且质量好.     

思茅松AFLP分子标记体系的建立

采用改进的SDS法,提取思茅松胚乳DNA,获得较高质量的DNA样品,用Eco RI和Pst I 2种限制性内切酶完全酶切后,再经过预扩和选扩,从82对引物中筛选出24对带型分布均匀、多态性高且分辨力强的引物。扩增产物经聚丙烯酰胺凝胶电泳后,可获得清晰指纹图谱,重复性较好。建立的思茅松AFLP体系可用于后续研究。     

拮抗链霉菌AFLP分析技术体系的研究

1材料与方法 1．1菌株供试拮抗链霉菌共10个菌株,均由该课题组分离自京郊菜田土壤和天然次生林土壤（表1）。     

拮抗链霉菌AFLP分析技术体系的研究(英文)

[Objective] The aim of this study was to investigate the preparation method and amplification system of antagonistic streptomyces DNA templates based on AFLP assays, and also provide a basis for the application of AFLP technology in the analysis of streptomyces or even actinomyces. [Method] The DNAs were extracted by the modified CTAB method and amplified by the Pst Ⅰ/Mse Ⅰ AFLP kit and its reaction system. The amplified products were analyzed by the denatured polyacrylamide gel electrophoresis. [Result] The genomic DNAs of ten antagonistic strains of Streptomyces were extracted and tested. The result of 0.8% agarose gel electrophoresis showed that the major DNA bands were clear without degradation and RNA residue, with the fragment sizes ranging from 37.64 to 40.86 Kb. By ultraviolet spectrophotometry, the OD260/OD280 values varying from 1.625 to 1.833 were obtained. Furthermore, the agarose gel electrophoresis of DNA products digested by Pst Ⅰ/Mse Ⅰ presented the dispersed fluorescent long band, which indicated that the enzymatic hydrolysis was fully carried out. The amplified bands of DNA templates by the screened three pairs of primers were clear with rich polymorphism. [Conclusion] The preparation method and amplification system of DNA template established in this study can be used in the AFLP analysis of Streptomyces.     

一种适用于动物与植物总DNA提取的方法——改良CTAB法

[目的]介绍一种简单、高效且能用于提取动物与植物的总DNA的方法。[方法]采用改良CTAB法,从24个花生品种嫩叶及小白鼠的肝、肺和肾中提取DNA,进行琼脂糖电泳和蛋白核酸分析仪检测及PCR扩增检验。[结果]所提取DNA电泳条带清晰,整齐均匀,OD260/OD280值介于1.77~1.83,用于PCR扩增获得理想效果。[结论]该研究介绍的改良CTAB法提取动物和植物的总DNA,满足开展PCR扩增的要求。     

大白菜基因组DNA的提取及AFLP反应体系的建立

1材料与方法1．1材料供试大白菜种质材料32份,分别由北京市农林科学院蔬菜研究中心品种资源库及蔬菜研究中心大白菜育种课题组和中国农业科学院蔬菜花卉研究所国家种质资源中期库提供。材料编号、品种名称、来源地见表1。     

雷公藤叶片基因组DNA不同提取方法比较

[目的]比较雷公藤叶片中基因组DNA的提取方法。[方法]采用常规CTAB法、改良CTAB法、常规SDS法、高盐低pH值法和一步法分别提取雷公藤叶片基因组DNA,利用琼脂糖凝胶电泳、紫外分光光度法测定样品OD260与OD280的比值以及RAPD扩增结果,判断所得DNA样品的纯度,并根据OD260值计算不同提取方法的DNA得率。[结果]改良CTAB法提取效果最佳,所提DNA的平均得率为219.3μg/g,OD260/OD280比值在1.869～1.903,用于RAPD-PCR均有较好的扩增结果。[结论]改良CTAB法是雷公藤叶片基因组DNA提取的最佳方法。     

A Method Suitable for Extracting Genomic DNA from Animal and Plant——Modified CTAB Method

[Objective] The study aimed to introduea a rapid and effective method that is suitable for extracting genomic DNA from animal and plant. [Method] The genomic DNAs were extracted from tender leaves of 24 peanut cuhivars and from the liver, lung and kidney of white mouse through the spe-cifically modified CTAB method. The DNAs were run on agarose gel, next detected by DNA/Protein analyzer. Finally PCR amplification was conducted to detect the quality of DNAs extracted using the modified CTAB method. [Result] The clear and orderly bands were observed in gel detection, and the val-ues of OD260/OD280 for DNAs extracted via modified CTAB method were between 1.77 - I. 83. The DNAs performed well in PCR amplification. [Conclu-sion] The DNAs extracted by modified CTAB method could satisfy the requirement of PCR amplification.     

A Method Suitable for Extracting Genomic DNA from Animal and Plant——Modified CTAB Method

[Objective] The study aimed to introduce a rapid and effective method that is suitable for extracting genomic DNA from animal and plant. [Method] The genomic DNAs were extracted from tender leaves of 24 peanut cultivars and from the liver,lung and kidney of white mouse through the specifically modified CTAB method. The DNAs were run on agarose gel,next detected by DNA/Protein analyzer. Finally PCR amplification was conducted to detect the quality of DNAs extracted using the modified CTAB method. [Result] The clear and orderly bands were observed in gel detection,and the values of OD260/OD280 for DNAs extracted via modified CTAB method were between 1.77-1.83. The DNAs performed well in PCR amplification. [Conclusion] The DNAs extracted by modified CTAB method could satisfy the requirement of PCR amplification.     

一种适用于动物与植物总DNA提取的方法——改良CTAB法

关于动物和植物基因组DNA的提取方法国内外有很多报道,但同一方法通常只能较好的从动物或植物提取总DNA。在前人报道的基础上,该研究对常用的CTAB法进行了适当修改,获得了一种从动物和植物中均能有效提取DNA的方法。     

一种适用于动物与植物总DNA提取的方法——改良CTAB法(英文)

[Objective] The study aimed to introduce a rapid and effective method that is suitable for extracting genomic DNA from animal and plant. [Method] The genomic DNAs were extracted from tender leaves of 24 peanut cultivars and from the liver,lung and kidney of white mouse through the specifically modified CTAB method. The DNAs were run on agarose gel,next detected by DNA/Protein analyzer. Finally PCR amplification was conducted to detect the quality of DNAs extracted using the modified CTAB method. [Result] The clear and orderly bands were observed in gel detection,and the values of OD260/OD280 for DNAs extracted via modified CTAB method were between 1.77-1.83. The DNAs performed well in PCR amplification. [Conclusion] The DNAs extracted by modified CTAB method could satisfy the requirement of PCR amplification.