水蛭ISSR-PCR反应体系的建立及优化

Establishment and optimization of ISSR-PCR reaction system for Hirudo nipponica

目的建立并优化水蛭ISSR-PCR反应体系及扩增程序,为水蛭进行遗传多样性研究提供依据。方法使用引物ISSR825,采用正交优化方法对影响PCR反应的Mg2+、dNTP、引物、Taq DNA聚合酶、模板DNA进行了体系优化,同时对引物的最佳退火温度进行了选择。结果在25μL总体积中,其中包括10×PCR缓冲液2.5μL、Mg2+2.0 mmol/L、dNTP0.25 mmol/L、引物0.8μmol/L、Taq DNA聚合酶2.5 U、模板DNA 2.0 ng/μL,最佳退火温度为49.7℃。结论所建立的最佳ISSR-PCR反应体系稳定可靠,可用于水蛭遗传多样性评价、不同种源鉴定及亲缘关系分析。

Objective To establish and optimize the ISSR-PCR reaction system and amplification procedure for Hirudo nipponica and to provide the basis for its genetic diversity research. Methods Using Primer ISSR825, the orthogonal test design was adopted to optimize the ISSR-PCR amplification system on H. nipponica in five factors （Mg2＋, dNTP, primer, Taq DNA polymerase, and template DNA） at four levels, and the suitable anneal temperature of the primer was selected. Results The suitable ISSR-PCR system （25 gL reaction volume） in Hi. nipponica included 10 x PCR buffer （2.5 μL）, Mg2＋ （2.0 mmol/L）, dNTP （0.25 μmmol/L）, primer （0.8 μmol/L）, Taq DNA polymerase （2.5 U）, and template DNA （2.0 ng/μL）; The suitable anneal temperature was 49.7 ℃. Conclusion The established and optimized ISSR reaction system is stable and reliable, which could provide the basis for the analysis of genetic diversity, germplasm resources, and genetic relationship ofH. nipponica.