文献类型：期刊文献

中文题名：多用途树种无患子ISSR-PCR体系建立与检测

英文题名：Detection and establishment of ISSR-PCR system for multipurpose tree species Sapindus mukorossi

作者：刁松锋[1,2] 邵文豪[1] 姜景民[1] 栾启福[1]

机构：[1]中国林业科学研究院亚热带林业研究所;[2]中国林业科学研究院经济林研究中心

年份：2015

卷号：35

期号：6

起止页码：899-904

中文期刊名：广西植物

外文期刊名：Guihaia

收录：北大核心:【北大核心2014】；CSCD:【CSCD_E2015_2016】；

基金：国家林业公益性科研专项(201404104;200804032);浙江省重大科技专项重点农业项目(2011C12015)

语种：中文

中文关键词：无患子;ISSR;PCR反应体系;正交设计

外文关键词：Sapindus mukorossi; ISSR; PCR reaction system; orthogonal design experiments;

分类号：S718.46;Q943

摘要：无患子(Sapindus mukorossi)是我国长江以南地区传统的重要绿化树种,其果皮富含皂苷,种仁富含油脂,为新型木本油料树种之一。为了获得基于KOD FX高保真DNA聚合酶试剂盒的无患子ISSR-PCR的最佳反应体系,该文采用正交优化设计相结合的方法,研究了引物浓度、d NTPs浓度、KOD FX酶、模板DNA浓度和Mg2+浓度对无患子ISSR-PCR反应体系的影响,并在此基础上对退火温度进一步优化,最终确立了适合无患子的ISSR-PCR反应的最佳体系和程序,即20μL PCR反应体系中,引物0.5μmol·L-1、d NTPs 0.05 mmol·L-1、KOD FX酶0.06 U·μL-1、模板DNA浓度1.0 ng·μL-1和Mg2+1.0 mmol·L-1。当以UBC841为引物时,PCR扩增程序为94℃预变性2 min,98℃变性10 s,48.6℃退火30 s,68℃延伸90 s,35个循环,68℃延伸7 min,4℃保存。这一优化的ISSR-PCR反应体系的建立,为无患子种质资源的遗传多样性、亲缘关系及遗传结构研究、种质创新与分子辅助育种等奠定了良好的基础。
Sapindus mukorossi is a traditional and important virescencetree species in the south part of China with rich saponins in peel and rich oil in seed. This tree species is one of the newly-developed woody oil species that were approved by the State Forestry Administration of China. For optimizing ISSR-PCR reaction system of S. mukorossi,based on KOD FX High-fidelity DNA Polymerase Kit,orthogonal design experiments were conducted. The main factors affecting ISSR-PCR amplification,such as suitable concentration of primer,d NTPs,KOD FX DNA polymerase,DNA template and 2 × PCR buffer for KOD FX were studied. Furthermore,the annealing temperature was optimized on the base of the above tests. An ideally ISSR-PCR reaction system was established,namely 25 μL reaction system containing primer 0. 5 μmol·L-1,d NTPs 0.05 mmol·L-1,KOD FX DNA polymerase 0. 06 U·μL-1、DNA template 1. 0 ng·μL-1and Mg2 ＋1. 0 mmol · L-1. The optimal PCR amplification program was： The profile of ISSR-PCR was an initial denaturation step for 2 min 94 ℃,followed by 35 cycles of 30 s at 98 ℃ for denaturation,30 s at 48. 6 ℃ for annealing,90 s at 68 ℃ for extension,finally extension at 68 ℃ for 7 min and holding the samples at 4 ℃. This optimized ISSRPCR reaction system would provide the basis for the analysis of genetic diversity,genetic structure,germplasm innovation and molecular assisted selection in S. mukorossi.