金柑CDDP-PCR反应体系优化研究

doi:10.3969/j.issn.1004-1524.2020.01.08

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (1): 65-71.DOI: 10.3969/j.issn.1004-1524.2020.01.08

金柑CDDP-PCR反应体系优化研究

陈燕丽^1,2, 唐志鹏¹, 欧克纬¹, 卢业飞², 张宇^1,2,3,*

1. 广西大学农学院,广西南宁 530004;
2. 广西壮族自治区亚热带作物研究所,广西南宁 530001;
3. 广西土壤肥料工作站,广西南宁 530007

收稿日期:2019-06-10 出版日期:2020-01-25 发布日期:2020-03-11
通讯作者: ^*张宇,E-mail:375900554@qq.com
作者简介:陈燕丽(1978—),女,广西平南人,硕士,农艺师,主要从事农业基础技术研究及推广等工作。E-mail:chengyanli9981@163.com
基金资助:
国家现代农业创新体系广西柑橘创新团队建设项目(2011A-06); 广西自然科学基金(2016GXNSFAA380164); 广西亚热带作物研究所基本科研业务费专项(桂热研201905)

Optimization of CDDP-PCR reaction system in Fortunella japonica

CHEN Yanli^1,2, TANG Zhipeng¹, OU Kewei¹, LU Yefei², ZHANG Yu^1,2,3,*

1. College of Agriculture, Guangxi University, Nanning 530004, China;
2. Guangxi Subtropical Crops Research Institute, Nanning 530001, China;
3. Guangxi Soil and Fertilizer Station, Nanning 530007, China

Received:2019-06-10 Online:2020-01-25 Published:2020-03-11

摘要/Abstract

摘要： 构建优化金柑CDDP-PCR反应体系并筛选适用于金柑CDDP-PCR分析的理想引物,为利用CDDP标记技术辅助种质鉴定以及分子育种等提供参考依据。以金柑基因组DNA为模板,采用正交优化试验设计方案,对dNTPs浓度、引物浓度、模板DNA用量、Mg²⁺浓度、Taq DNA聚合酶浓度设计5因素4水平试验,采用极差分析法、方差分析法和Duncan多重比较法对试验结果进行分析。结果表明:利用供试的5个金柑品种验证试验效果,21条CDDP通用引物均可扩增出多态性条带。最佳反应体系为:dNTPs浓度为0.15 mmol·L^-1,引物浓度为1.0 μmol·L^-1,模板DNA用量为120 ng,Mg²⁺浓度为1.5 mmol·L^-1,Taq DNA聚合酶浓度为0.25 U,剩余体积用ddH₂O补足至20 μL。各因素对反应体系影响从大到小依次为dNTPs>引物>模板DNA>Mg²⁺>Taq DNA聚合酶。

关键词: 金柑, CDDP, 反应体系, 优化

Abstract: To construct and optimize the CDDP-PCR reaction system of Fortunella japonica and select the ideal primers for the CDDP-PCR analysis of Fortunella japonica, so as to provide reference basis for using CDDP marker technology to assist germplasm identification and molecular breeding, the DNA of Fortunella japonica genome was used as the template, and the orthogonal optimization test design scheme was adopted to design the 5 factor and 4 level test for dNTPs concentration, primer concentration, template DNA dosage, Mg²⁺ concentration and Taq DNA polymerase concentration. Range analysis method, variance analysis method and Duncan multiple comparison method were used to analyze the test results. The results showed that polymorphic bands could be amplified from 21 common primers of CDDP using 5 Fortunella japonica varieties. The optimal reaction system was as follows: DNA dosage 120 ng, Mg²⁺ concentration 1.5 mmol·L^-1, Taq DNA polymerase concentration 0.25 U, primer concentration 1.0 μmol·L^-1, dNTPs concentration 0.15 mmol·L^-1, and the remaining volume was supplemented by ddH₂O to 20 μL. The influences of various factors on the reaction system from the largest to the smallest were dNTPs>primer>template DNA>Mg²⁺>Taq DNA polymerase.

Key words: Fortunella japonica, CDDP, reaction system, optimization

中图分类号:

S666.1

陈燕丽, 唐志鹏, 欧克纬, 卢业飞, 张宇. 金柑CDDP-PCR反应体系优化研究[J]. 浙江农业学报, 2020, 32(1): 65-71.

CHEN Yanli, TANG Zhipeng, OU Kewei, LU Yefei, ZHANG Yu. Optimization of CDDP-PCR reaction system in Fortunella japonica[J]. , 2020, 32(1): 65-71.

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金柑CDDP-PCR反应体系优化研究

Optimization of CDDP-PCR reaction system in Fortunella japonica

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