CAJ | 학술논문

以‘兰州百合’为试材,采用CTAB法提取百合嫩叶 DNA,对‘兰州百合’ISSR-PCR反应体系的Taq酶、dNTP、引物和模板DNA进行4个因素4个水平优化试验,通过聚丙烯酰胺凝胶电泳结果分析,最终获得的最佳反应体系为：25μL反应体系中含Taq DNA聚合酶0.5 U,dNTP 0.3 mmol/L,引物0.5μmol/L,模板 DNA 60 ng,10×Buffer(20 mmol/L Mg2+plus)2.5μL,不足部分用 ddH2 O补足.在获得最佳反应体系的基础上确定了引物最佳退火温度为53℃,最佳循环次数45次.该优化体系以 UBC873作为引物对18份‘兰州百合’材料DNA进行 IS-SR-PCR扩增,结果显示该优化体系具有较高的扩增稳定性.
이‘란주백합’위시재,채용CTAB법제취백합눈협 DNA,대‘란주백합’ISSR-PCR반응체계적Taq매、dNTP、인물화모판DNA진행4개인소4개수평우화시험,통과취병희선알응효전영결과분석,최종획득적최가반응체계위：25μL반응체계중함Taq DNA취합매0.5 U,dNTP 0.3 mmol/L,인물0.5μmol/L,모판 DNA 60 ng,10×Buffer(20 mmol/L Mg2+plus)2.5μL,불족부분용 ddH2 O보족.재획득최가반응체계적기출상학정료인물최가퇴화온도위53℃,최가순배차수45차.해우화체계이 UBC873작위인물대18빈‘란주백합’재료DNA진행 IS-SR-PCR확증,결과현시해우화체계구유교고적확증은정성.
In order to get reliable and repeatable reaction system of ISSR-PCR amplification for‘Lanzhou lily’,we extracted its genomic DNA from young leaves by the methods of modified CTAB,then we optimized the ISSR-PCR reaction system at 4 levels and 4 factors,Taq DNA polymerase,dNTP,prim-ers,DNA template.The results of polyacrylamide gel electrophoresis showed that the best reaction system was 25μL reaction solution,which contained 0.5 U Taq DNA polymerase,dNTP 0.3 mmol/L,0.5μmol/L primers,60 ng template DNA and ddH2 O.We found that 53 ℃ was the best PCR annealing temperature and 45 cycles was the best cycle times.The stabilization of the optimized reaction system was tested by u-sing the primer UBC873 on 18 ‘Lanzhou lily’,the results showed that the optimized reaction system had the high stability .