CAJ | 학술논문

探讨了大白菜EST-SSR反应体系的优化,利用优化的体系筛选部分引物.采用L_(16)(4~4)正交设计,研究各主要参数的适宜浓度,建立适合大白菜EST-SSR反应的最佳体系.结果表明,各因素不同水平浓度对扩增结果均有一定影响,其中以引物浓度影响最大,其次是dNTP和Taq DNA聚合酶,最小是模板DNA用量.优化后的最佳体系(20μL)为:引物浓度1.0μmol/L,dNTPs0.25mmol/L,Taq酶0.5U,模板DNA 70ng.利用优化的体系,根据大白菜EST序列设计引物126对,分别以2对抗、感病毒病的大白菜材料的基因组DNA为模板进行扩增,筛选出至少在一份材料中扩出多态性的引物19对.该优化体系可用于大白菜EST-SSR扩增,初步筛选的引物可用于大白菜病毒病分子标记的进一步研究.
탐토료대백채EST-SSR반응체계적우화,이용우화적체계사선부분인물.채용L_(16)(4~4)정교설계,연구각주요삼수적괄의농도,건립괄합대백채EST-SSR반응적최가체계.결과표명,각인소불동수평농도대확증결과균유일정영향,기중이인물농도영향최대,기차시dNTP화Taq DNA취합매,최소시모판DNA용량.우화후적최가체계(20μL)위:인물농도1.0μmol/L,dNTPs0.25mmol/L,Taq매0.5U,모판DNA 70ng.이용우화적체계,근거대백채EST서렬설계인물126대,분별이2대항、감병독병적대백채재료적기인조DNA위모판진행확증,사선출지소재일빈재료중확출다태성적인물19대.해우화체계가용우대백채EST-SSR확증,초보사선적인물가용우대백채병독병분자표기적진일보연구.
The aim of this paper is to optimize the EST-SSR system on heading Chinese cabbage,and select a certain amount of primer pairs using the optimized system.L_(16)(4~4) orthogonal design is used in the system optimization,and the concentrations of the main factors are studied to build the best system.The results show that the different levels of each factor have some effects on the results of EST-SSR,with the effect of the primer concentration being the greatest,those of dNTP and Taq DNA polymerase the next,and that of the concentration of DNA template the minimal.The optimized system (20μL) is:2.0μL 10×PCR Buffer with Mg~(2+),70ng DNA template,250μmol/L dNTP,1.0μmol/L primer,and 0.SU Taq DNA polymerase.One hundred and twenty-six primer pairs are designed according to the EST sequences of heading Chinese cabbage,and EST-SSR PCR is amplified by using the above primer pairs and optimized system,with nineteen primer pairs being selected based on their reproducible and polymorphism,which could be used for further research.