CAJ | 학술논문

本研究以仁用杏丰仁为试材,采用经改良的CTAB法提取实验材料幼叶基因组DNA.利用正交设计法,从Mg~(2+)浓度、dNTPs浓度、引物浓度、Taq/DNA聚合酶及模板DNA用最5种因素4水平出发,构建和优化仁用杏ISSR-PCR反应体系,并采用直观分析和方差分析相结合的方法分析正交实验结果,最终建立了仁用杏ISSR-PCR最佳反应体系总体积20μL:1.5 U Taw DNA聚合酶、2.0 mmol/L MgCl_2、0.15 mmol/L dNTPs、0.35μmol/L引物、30～60 ng模板DNA.在优化体系的基础上筛选出 13个扩增稳定、多态性丰富的ISSR引物,并通过梯度PCR试验,确定每个引物的最适退火温度.经对最佳反应体系和程序进行了验证,结果显示该体系具有扩增稳定性.
본연구이인용행봉인위시재,채용경개량적CTAB법제취실험재료유협기인조DNA.이용정교설계법,종Mg~(2+)농도、dNTPs농도、인물농도、Taq/DNA취합매급모판DNA용최5충인소4수평출발,구건화우화인용행ISSR-PCR반응체계,병채용직관분석화방차분석상결합적방법분석정교실험결과,최종건립료인용행ISSR-PCR최가반응체계총체적20μL:1.5 U Taw DNA취합매、2.0 mmol/L MgCl_2、0.15 mmol/L dNTPs、0.35μmol/L인물、30～60 ng모판DNA.재우화체계적기출상사선출 13개확증은정、다태성봉부적ISSR인물,병통과제도PCR시험,학정매개인물적최괄퇴화온도.경대최가반응체계화정서진행료험증,결과현시해체계구유확증은정성.
In this research, a kind of apricot of kernel-consuming named Fengren was used to be as experiment materials, and an improved method of CTAB was applied to extract the genomic DNA from its leaf spires. We completed the construction and optimization of ISSR-PCR systerm in apricot of kernel-consuming by employing L_(16)(4~5) orthogonal-design with four levels and five factors, which are Mg~(2+), dNTPs, primers, Taq DNA polymerases, and template DNA, then the results of ISSR-PCR reaction and orthogonal design were analyzed by the combining method of intuitive analysis and variance analysis, finally, optimistic ISSR-PCR system in apricot of kemel-consuming was established in 20μL reaction solution, contained 1.5μL Tat/DNA polymerase, 2.0 mmol/L MgCl_2, 0.15 mmol/L dNTPs,0.35 μmol/L primers and 30～60 ng template DNA. Thirteen primers with stable amplification and rich polymorphism were obatined based on the optimal PCR reaction system, and across gradient PCR experiment to decide the optional annealing temperature of every one primes. Verification of the optimistic ISSR-PCR reaction system and amplification procedures showed that this amplification system was stable.