濒危植物峨眉野连ISSR反应体系的建立与优化

Establishment and optimization of ISSR reaction system for endangered plant Coptis omeiensis

关键词:  峨眉野连  ISSR  建立  优化

Key words:  Coptis omeiensis  ISSR  establishment  optimization

摘要:

针对峨眉野连ISSR的反应特点,建立稳定可靠的ISSR-PCR分子标记反应体系,为进一步研究峨眉野连的种质资源遗传多样性奠定基础。通过筛选引物并设定影响峨眉野连ISSR-PCR反应诸因子的不同梯度,检测其不同反应体系的扩增效果,分析非特异性条带的产生原因并进行条件优化,建立峨眉野连ISSR-PCR稳定可靠的反应体系。首次建立了可用于峨眉野连ISSR-PCR分析的最适宜的反应体系:25μL PCR反应体系中,内含1×PCR Buffer,1.5 mmol/LMg²⁺,200 μmol/L dNTP,0.3 μmol/L引物,80 ng模板,1.0 U Taq DNA聚合酶。扩增程序为94 ℃预变性5 min,然后进行35个循环:94 ℃变性30 s,(据不同引物的退火温度)复性60 s,72 ℃延伸90 s,循环结束后72 ℃延伸7 min,4 ℃保存。所建立的峨眉野连ISSR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力强、重复性好等特点,可以较好地应用于峨眉野连的种质资源多样性及居群鉴别的研究。

Abstract:

In order to study the genetic diversity of Coptis omeiensis germplasm,ISSR-PCR system of C.omeiensis was established and optimized according to the characters of it. The effects of ISSR-PCR was examined by selecting primers and designing different concentrations of the factors in the ISSR,the reliable systems for C.omeiensis populations researching was established by analyzing the reasons for occurrence of differential bands and optimizing reaction conditions. The optimal ISSR-PCR system in C.omeiensis was established for the first time,that is,25 μL amplification reactions system containing 1×PCR Buffer,1.5 mmol/L Mg²⁺,200 μmol/L dNTP,0.3 μmol/L primer,80 ng template,1.0 U Taq DNA polymerase.The optimal amplified procedure was as follows:after a pre-denaturing of 5 min at 94 ℃,35 cycles were performed with denaturing of 30 s at 94 ℃,annealing of 1min due to denaturing temperature of different primer,extension of 1.5min at 72 ℃,a final extension step of 7 min at 72 ℃ and hold at 4 ℃. The ISSR-PCR systems,which were established in this paper for studying C.omeiensis,could provide clear reliable abundant polymorphisms molecular markers and were proved suitable for germplasm resource studying and identification of C.omeiensis.