白木香基因组DNA的提取及ISSR反应条件的优化

Extraction of Genomic DNA and Optimization of ISSR Reaction Condition for Aquilaria sinensis(Lour.) Gilg

[目的]以白木香为试验材料，研究基因组DNA提取方法，并优化ISSR-PCR反应体系。[方法]利用快速提取仪和微量液氮法提取DNA，并对ISSR-PCR反应体系进行单因素筛选。[结果]微量液氮法提取的DNA质量好于快速提取仪，但2种方法得到的DNA对后续ISSR研究没有明显的差异。同时，建立了最适的白木香ISSR-PCR体系，即25出PCR反应体积中，1×PCR buffer，2．0mmol／LMgCl2，4ng／ul模板DNA，200umol／LaNTes，1．1 U Taq DNA聚合酶，0．4umol／L引物。最佳扩增程序为：94℃预变性5min，然后进行45个循环，94℃变性45s，复性温度根据各引物的TM值略低1—2℃，45s，72℃延伸75s，循环结束后72℃延伸7min。[结论]优化系统的建立为进一步利用ISSR分子标记技术进行白木香遗传多样性研究提供了基础。

[ Objective ] The extraction method of genomic DNA and optimization of ISSR-PCR reaction condltion for Aquilaria sinensis （Lour.） Gilg were studied. [ Method] Two different methods including grinded with BIO101 Fastprep and trace liquid nitrogen methed were used to extract the genomic DNA. Single factor expriment was carried out to optimize ISSR-PCR reaction condition. [ Result] The quality of extracted DNA with BIO101 Fastprep was better than that of trace liquid nitrogen method, but there was no obvious distinction between the results of ISSR-PCR with different methods. The reaction system and amplified procedure suitable for A. sinensis were as follows： 25 gl amplification reactions system containing 1 × PCR buffer, 2.0 mmol/L MgCl2, 4 ng/p.1 template DNA, 200umol/L dNTPs, 1.1 U Taq DNA polymerase and 0.4umol/L primer. The optimal amplified procedure was as follows： after a pre-denaturing of 5 min at 94 ℃, 45 cycles were performed with denaturing of 45 s at 94 ℃, annealing of 45 s due to 1 - 2 ℃ lower than denaturing temperature of different primer, extension of 75 s at 72 ℃, a fmal extension step of 7 vain at 72 ℃. [Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of A. sinensis using ISSR molecular marker.