摘要
利用5因素4水平的正交实验探讨了各因素的不同水平对峨眉金线莲ISSR-PCR反应结果的影响,建立了稳定且重复性好的峨眉金线莲ISSR-PCR反应体系,即在25μL体系中含:40 ng DNA模板、0.4μmol·L^(−1)引物、0.4 mmol·L^(−1)dNTPs、2.0 mmol·L^(−1)Mg^(2+)、0.8 U Taq DNA聚合酶和2.0μL 10×Buffer。同时为了确定最适Tm(退火温度),进行了梯度PCR试验筛选,最终优化和确立了适合峨眉金线莲的ISSR-PCR实验反应体系。
The effects of different levels of factors on ISSR-PCR reaction results of Anoectochilus omeiensis were discussed by orthogonal design with 5 factors and 4 levels,and a stable and reproducible ISSR-PCR reaction system of Anoectochilus omeiensis was established.That is,the 25μL system contained 40ng DNA template,0.4μmol/L primer,0.4 mmol/L dNTPs,0.8 U Taq DNA polymerse,2 mmol/L Mg^(2+),and 2.0μL 10×BufferμL in 25μL reaction volume.At the same time,in order to determine the optimum Tm(annealing temperature),gradient PCR test was carried out,and finally ISSR-PCR experimental reaction system suitable for Anoectochilus omeiensis was optimized and established.
作者
谢孔平
杨适
毛冀
鲁松
XIE Kongping;YANG Shi;MAO Ji;LU Song(Sichuan Provincial Institute of Natural Resources Science,Chengdu 610015,China;Wild Plants Sharing and Service Platform of Sichuan Province,Chengdu 610015,China;Emeishan Biodiversity Observation and Research Station of Sichuan Province,Emeishan 610041,China)
出处
《四川林业科技》
2022年第4期76-82,共7页
Journal of Sichuan Forestry Science and Technology
基金
四川省重点研发项目(2018SZ0277
2018SZ0294)
四川省科技基础条件平台项目(2019JDPT0028)
2020野生植物四川省科技资源共享服务平台
中国科学院山地生态恢复与生物资源利用重点实验室-生态恢复与生物多样性保育四川省重点实验室开放课题项目(kxysws1607)。
关键词
峨眉金线莲
ISSR
正交试验
Anoectochilus emeiensis
ISSR
Orthogonal experimental design
