CAJ | 학술논문

为优化羽衣甘蓝(Brassica oleracea var. acephala)SSR反应体系,并利用优化的体系进行引物的筛选。采用单因素完全随机试验筛选各反应因素的最佳水平,并采用L16(45)正交设计进行试验优化,建立了羽衣甘蓝SSR-PCR最佳反应体系。结果表明,各因素不同水平浓度对扩增结果均有一定影响,羽衣甘蓝基因组DNA的最佳SSR反应体系为DNA模板量(50 ng/μL)1.0μL,10×PCR Buffer 1.0μL,引物量(2.0μmol/L)上下游各3.0μL,dNTPs(2.5 mmol/L)0.8μL,Taq聚合酶(5 U/μL)0.2μL,ddH2O 1.0μL,总体积10μL。检测了优化体系的稳定性,利用优化的反应体系对芸薹属植物20对SSR引物进行扩增并用聚丙烯酰胺凝胶电泳检测,在所选用的20对引物中,18对扩增出了清晰的条带,11对具有特异扩增条带,多态性引物比率为61%,验证了该体系可用于羽衣甘蓝SSR-PCR扩增和SSR引物的筛选。
위우화우의감람(Brassica oleracea var. acephala)SSR반응체계,병이용우화적체계진행인물적사선。채용단인소완전수궤시험사선각반응인소적최가수평,병채용L16(45)정교설계진행시험우화,건립료우의감람SSR-PCR최가반응체계。결과표명,각인소불동수평농도대확증결과균유일정영향,우의감람기인조DNA적최가SSR반응체계위DNA모판량(50 ng/μL)1.0μL,10×PCR Buffer 1.0μL,인물량(2.0μmol/L)상하유각3.0μL,dNTPs(2.5 mmol/L)0.8μL,Taq취합매(5 U/μL)0.2μL,ddH2O 1.0μL,총체적10μL。검측료우화체계적은정성,이용우화적반응체계대예대속식물20대SSR인물진행확증병용취병희선알응효전영검측,재소선용적20대인물중,18대확증출료청석적조대,11대구유특이확증조대,다태성인물비솔위61%,험증료해체계가용우우의감람SSR-PCR확증화SSR인물적사선。
The SSR system of Brassica oleracea var. acephala was optimized by screening primers. The single factor with ran-dom design was used to optimize the SSR-PCR amplification system and the L16 (45) orthogonal design was used. Results showed that different levels of each factor had different effects on PCR. The optimal system was DNA template (50 ng/μL) 1.0 μL, 10×PCR Buffer1.0 μL, 2.0 μmol/L upstream primer and downstream 3.0 μL each, dNTPs(2.5 mmol/L)0.8 μL, Taq DNA polymerase (5U/μL) 0.2 μL and ddH2O 1.0 μL. Twenty pairs of primers were used to test the optimized system by polyacrylamide gel electrophoresis. Eighteen pairs of primers had clear bands and eleven pairs of primers were with polymor-phic, with the percentage of polymorphism of 61%. The optimized system could be used for SSR-PCR.