仁用杏ISSR分析体系的正交优化  

Optimization of an ISSR Analysis System of Apricot of Kernel-Consuming Based on Orthogonal Design

作　　者：白锦军[1] 魏安智[1] 王佳[1] 张亚转[1] 

机构地区：[1]西北农林科技大学生命学院,杨凌712100

出　　处：《分子植物育种》2009年第6期1237-1244,共8页Molecular Plant Breeding

基　　金：国家十一五科技支撑项目(2006BAD18B0201);陕西省重大科技专项计划项目(2006kz09-G6)共同资助

摘　　要：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Mg2+浓度、dNTPs浓度、引物浓度、TaqDNA聚合酶及模板DNA用量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:1.5UTaqDNA聚合酶、2.0mmol/LMgCl2、0.15mmol/LdNTPs、0.35μmol/L引物、30～60ng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。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 L16（45） orthogonal-design with four levels and five factors,which are Mg2＋,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 kernel-consuming was established in 20 μL reaction solution,contained 1.5 μL Taq DNA polymerase,2.0 mmol/L MgCl2,0.15 mmol/L dNTPs,0.35 μmol/L primers and 30-60 ng template DNA.Thirteen primers with stable amplification and rich polymor-phism 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.

关 键 词：仁用杏 ISSR 正交优化 

分 类 号：S662.2[农业科学—果树学] TS452.3[农业科学—园艺学]