山竹子基因组DNA提取及SRAP反应体系优化

Genome DNA Extraction and SRAP Reaction System Optimization of Garcinia mangostana

采用改良CTAB法提取山竹子基因组DNA,并以me1和em3正反向引物组合对山竹子进行了SRAP体系的优化。结果表明：使用CTAB free缓冲液进行前处理可去除大部分的杂质,获得的DNA纯度较高,OD260/OD280均在1.7~1.9之间,RNA消化彻底,DNA无明显降解,进行SRAP分析条带清晰,多态性好。在25μL反应体系中,5种主要成分的适宜浓度或用量分别是：Taq DNA聚合酶0.5 U,Mg2＋2.0 mmol.L-1,dNTPs 0.15~0.2 mmol.L-1,模板DNA 10~50 ng,引物0.6~0.8μmol.L-1。该体系适用于山竹子SRAP分析,进行遗传图谱构建、基因定位与克隆、比较基因组学、遗传多样性、cDNA指纹图谱等方面的研究。

The modified CTAB method was studied to extract genomic DNA from Garcinia rnangostana, and the optimal system of SRAP analysis was established with the primer combination of me1 and em3. The results showed that the modified protocol could obtain high quality DNA, which reached perfect values of OD260/OD280 （1. 7 - 1. 9）, free RNA contamination and DNA in good integralities. SRAP-PCR analysis with the primer combination of mel and em3 further confirmed the DNA extracted by modified protocol might be suitable for genetic analysis, which could generate recordable polymorphic banding patterns, and the optimum concentrations of five important ingredients in 25μL reaction system were.. Taq DNA polymerase 0.5 U, Mg2＋ 2.0 mmol ·L^-1, dNTPs 0.15-0.2 mmol · L^-1, template DNA 10- 50 ng and primers 0. 6- 0. 8 μmol · L^-1. This reaction system can be used for SRAP of Garcinia mangostana in genetics mapping, gene tagging cloning, comparative genomics, genetic diversify and eDNA fingerprinting.