峨眉金线莲ISSR反应体系的建立与优化  

Establishment and Optimization of ISSR-PCR Reaction System of Anoectochilus emeiensis

作　　者：谢孔平 杨适[1] 毛冀[1] 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)

机构地区：[1]四川省自然资源科学研究院,四川成都610015 [2]野生植物四川省科技资源共享服务平台,四川成都610015 [3]峨眉山生物多样性四川省野外科学观测研究站,四川峨眉山610041

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)。

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.

关 键 词：峨眉金线莲 ISSR 正交试验 

分 类 号：S567.239[农业科学—中草药栽培]