福建道地药材枇杷叶ISSR反应体系的建立与优化

Establishment and Optimization of ISSR Reaction System for Eriobotryae Folium from Fujian

关键词:  枇杷叶  简单重复间隔序列  反应体系  优化

Key words:  Eriobotryae Folium  ISSR  reaction system  optimization

摘要:

目的 建立适于福建产枇杷叶的ISSR分析的PCR反应体系，筛选适宜引物，并确定最佳退火温度。方法 改良的CTAB法提取嫩叶中DNA，通过单因子实验分别找出合适的ISSR-PCR反应条件，利用梯度PCR确定引物最佳退火温度。结果 适于枇杷叶ISSR最佳的反应体系为：总反应体积为25 μL，其中模板DNA 60 ng，Taq DNA聚合酶0.9 U，引物浓度0.4 μmol·L^-1，dNTP浓度200 μmol·L^-1，10×Buffer(含Mg²⁺) 2.5 μL，加入灭菌去离子水至25 μL。扩增程序为：预变性94 ℃ 7 min，变性94 ℃ l min，复性最佳退火温度1 min，延伸72 ℃ 1 min，35个循环，72 ℃延伸10 min。在100条引物中筛选出14条扩增条带清晰且条带数目较多的引物，最佳退火温度为48~58 ℃(因引物不同而异)。 结论 建立了枇杷叶ISSR最佳反应体系，筛选出14条引物并确定了最佳退火温度，为应用ISSR技术鉴定枇杷叶的种质资源和遗传多样性研究奠定了基础。

Abstract:

OBJECTIVE To establish and optimize the ISSR-PCR reaction system for Eriobotryae Folium; to screen the suitable primers and determine their optimal annealing temperatures. METHODS Genomic DNA was extracted by CTAB method from Eriobotrya leaves. The main influencing elements in different levels were tested by single factor experiment. The gradient PCR was used to determine the optimal annealing temperatures of each selected primer. RESULTS The optimized PCR reaction system: total response volume 25 μL, including 60 ng template DNA, DNA polymerases dosage for 0.9 U, primer eventual concentration of 0.4 mol·L^{-1, dNTP eventual concentration was 200 μmol·L-1, 10×Buffer(including Mg2+) for 2.5 μL, sterilization deionized water to 25 μL. The optimal amplified procedure was as follows: after a pre-denaturing of 7 min at 94 ℃, 35 cycles were performed with denaturing of 1 min at 94 ℃, annealing of 1 min according to denaturing temperature of different primers, extension of 1 min at 72 ℃, a final extension step of 10 min at 72 ℃. Forteen primers which showed clearer and more bands from 100 primers were selected and the optimal annealing temperatures were 48-58 ℃(changed among different primers). CONCLUSION The stable and reproducible optimal ISSR-PCR reaction system and suitable annealing temperatures of 14 selected primers from 100 are established for Eriobotryae Folium which had laid the good foundation for ISSR analysis on studies of germplasm resources identification and genetic diversity.}