白木香基因组DNA的提取及ISSR反应条件的优化

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25lμPCR反应体积中,1×PCR buffer,2.0 mmol/L MgCl2,4ng/μl模板DNA,200μmol/L dNTPs,1.1 UTaqDNA聚合酶,0.4μmol/L引物。最佳扩增程序为:94℃预变性5 min,然后进行45个循环,94℃变性45 s,复性温度根据各引物的TM值略低1~2℃,45 s,72℃延伸75 s,循环结束后72℃延伸7 min。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。

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

Objective] The extraction method of genomic DNA and optimization of ISSR-PCR reaction condition for Aquilaria sinensis(Lour.) Gilg were studied. Method] Two different methods including grinded with BIO101 Fastprep and trace liquid nitrogen method 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 μl amplification reactions system containing 1×PCR buffer,2.0 mmol/L MgCl2,4 ng/μl template DNA,200 μmol/L dNTPs,1.1 U Taq DNA polymerase and 0.4 μmol/L primer.The optimal amplified procedure was as follows: after a predenaturing 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 final extension step of 7 min at 72 ℃.Conclusion] The optimal system could provide a favorable basis for further study on genetic diversity of A.sinensis using ISSR molecular marker.