均匀设计与正交设计优化海岛棉RGA-PCR体系

Optimization of RGA-PCR System of Sea-island Cotton by Uniform Design and Orthogonal Design

采用均匀设计和正交设计对影响海岛棉RGA体系Mg^(2+)、dNTP、引物以及Taq DNA聚合酶浓度等因素进行了均匀优化和正交优化试验后建立了适合于海岛棉RGA的反应体系:在10μL反应体系中1×Buffer,Mg^(2+)2.5 mmol/L,dNTP 0.25 mmol/L,引物浓度1.0μmmol/L,Taq酶0.375 U/μL,DNA 20 ng。各因素对扩增反应结果均有不同影响,其中以Mg^(2+)浓度影响最大,Taq DNA聚合酶的影响最小。运用该体系对海岛棉材料进行验证,证明该体系稳定可靠,并从22对RGA引物组合中筛选出扩增条带清晰、多态性丰富的10对引物。这一体系的建立及多态性引物的筛选为今后利用RGA标记技术进行海岛棉枯萎病研究提供了科学依据。

Uniform design and orthogonal design were applied to optimize RGA- PCR system of seaisland cotton. Four influencing factors on RGA - PCR, including Mg^2 ＋ , d NTP, primer and Taq DNA polymerase concentration were tested using uniform design and orthogonal design. A suitable RGA- RCR system for sea - island cotton was established as follows： each 10ul RGA - PCR amplification reaction solution was consisted of 1 × Buffer 2.5 mmol/L Mg^2＋ , 0.25 mmol/L dNTP, 1.0 μmmol/L primer, 0. 375U Taq DNA polymerase and 20ng template DNA. Each factor had a different effect on the results of PCR. Mg2 ＋ content ration had the greatest effect and Taq DNA polymerase had the least effect. The optimized RGA - PCR system was tested on seaisland cotton andshown to be steady and reliable. 10 primer combinations were selected with abundant polymorphism from 22 primer combinations. The optimized RGA - PCR system and polymorphism primer combinations could be applied to research of sea- island cotton.