枣cDNA-AFLP技术体系建立与优化

建立了枣(Ziziphus jujuba Mill.)cDNA-AFLP分析的优化体系和反应程序。提取获得的总RNA以人工合成的Oligo(dT)18为引物,利用鼠源反转录酶(M-MLV RT)合成cDNA第1链,合成双链cDNA后用限制性内切酶EcoRⅠ(识别6个碱基位点)与MseⅠ(识别4个碱基位点)酶切,酶切连接后,使用与接头序列互补的预扩引物对cDNA片段池进行PCR扩增,扩增后的cDNA池定量至1 ng/μL,作为选择性扩增的模板,优化得到的选扩反应体系为:20μL的反应体系中含5μL模板,2μL 10×PCR buffer,2μL MgCl2(25 mmol/L),EcoRⅠ特异性引物(50 ng/μL)1μL,MseⅠ特异性引物(50 ng/μL)1μL,10 mmol/L dNTP0.4μL,5 UTaqDNAPolymer-ase 0.12μL。选扩程序:94℃预变性2 min;94℃变性30 s,65℃退火30 s(每个循环降0.7℃),72℃延伸1 min,13个循环;94℃变性30 s,56℃退火30 s,72℃延伸1 min,31个循环;72℃7 min。

Establishment and optimization of cDNA-AFLP system in Ziziphus jujuba Mill.

An optimized cDNA-AFLP system and the PCR program suitable for Chinese jujube(Ziziphus jujuba Mill.) were established.After being digested using restriction endonuclease EcoR I and Mse I,and jointed with the relevant anchor,the cDNA pool was used as primary template for pre-amplification.The pre-amplification was diluted to the concentration of 1 ng/μL,which was used as the template for the selective amplification.Through groping and optimizing the reaction of AFLP selective amplification,the optimum system was established as follows: 5 μl template in 20 μL reaction systems,2 μL 10×PCR buffer,2 μLMgCl2(25 mmol/L),10 mmol/L dNTP 0.4 μL,5 U Taq DNA polymerase 0.12 μL,EcoR I selective primer(50 ng/μL)1 μL,MseI selective primer(50 ng/μL)1 μL.PCR program was that the first cycle performed as 2 min at 94 ℃ to predenature the template,then keep 94 ℃ for 30 s to denature,65 ℃ for 30 s to anneal(dropped 0.7 ℃ next cycle),72 ℃ for 1 min to extend,preceding process for 13 cycles;then 94 ℃ for 30 s to denature,56 ℃ for 30 s to anneal,72 ℃ for 1min to extend for 31 cycles and a final extension at 72 ℃ for 7 min after the last cycle.