农业研究与应用2025，Vol.38Issue(1)：9-18,10.DOI:10.20191/j.cnki.2095-0764.2025.01.002
金花菌ISSR-PCR反应体系的建立与优化
Establishment and Optimization of ISSR-PCR Reaction System for Golden Flower Fungus
摘要
Abstract
[Objective]Golden flower fungus is a kind of potential probiotic fungus in black tea.It can give tea a unique taste,and has a variety of health care effects such as supplementing vita-mins and minerals for human,producing saliva to quench thirst,breaking down greasiness,reduc-ing fat and losing weight,and is one of the important indexes to evaluate the quality of Fuzhuan tea and Liubao tea.In this study,the genetic diversity and structure of golden flower fungus were ana-lyzed,and the ISSR-PCR reaction system of golden flower fungus was established and optimized,aiming to provide a method for further systematic study of the genetic diversity and intraspecific and interspecific relationships of golden flower fungus using ISSR molecular marker technology.[Meth-od]In this study,the DNA of Aspergillus cristatum was used as a template,single-factor experi-ments were used to optimize the amount of template DNA,primer,PCR Mix and ISSR-PCR cycle number that affect the ISSR-PCR reaction system.The stability of the reaction system was verified by the strains of four golden flower fungi species,A.cristatum,A.chevalieri,A.pseudoglaucus and A.cibarius.[Result]An ISSR-PCR system for golden flower fungus was established,which con-sisted of 50 ng/μL template DNA 0.3 μL,10 μmol/μL primer 0.8 μL,2 SanTaq PCR Mix amount 9.0 μL,ddH2O 9.9 μL,and their total volume was 20 μL.The ISSR-PCR reaction conditions were pre-denaturation at 94.0℃for 5 min,denaturation at 94.0℃for 30 s,annealing for 30 s,exten-sion at 72.0℃for 2 min,with a total of 35 cycles;extension at 72.0℃for 10 min.The optimal ISSR-PCR reaction system was obtained through verifying four different golden flower fungi spe-cies stains,which showed its high polymorphism and repeatability.The optimized system was used to select 11 ISSR primers with high polymorphism and repeatability that could be used for genetic di-versity analysis of golden flower fungus and their annealing temperatures were also determined.[Conclusion]This study established and optimized the ISSR-PCR reaction system of golden flower fungus,and the results showed high polymorphism and repeatability,proving that it is a new meth-od for the genetic diversity analysis of golden flower fungus.关键词
金花菌/ISSR-PCR/单因素实验/反应体系Key words
Golden flower fungus/ISSR-PCR/single factor test/reaction system分类
生物学引用本文复制引用
吴凤,张莉娟,李冬桂,秦玉燕,罗义灿,陈羽烨,李鸿,吕丽兰..金花菌ISSR-PCR反应体系的建立与优化[J].农业研究与应用,2025,38(1):9-18,10.基金项目
广西自然科学基金项目(2022GXNSFBA035445) （2022GXNSFBA035445）
广西农业科学院基本科研业务专项(桂农科2024YP121,桂农科2021YT148) （桂农科2024YP121,桂农科2021YT148）
广西农业科学院科技发展基金项目(桂农科2020YM140). （桂农科2020YM140）
