旱半夏ISSR-PCR反应体系的优化  

Optimization of ISSR-PCR Reaction System in Pinellia ternata

作　　者：喻娜 唐宇翀[2] 刘勇 戴炜 Yu Na;Tang Yuchong;Liu Yong;Dai Wei(Department of Medicine,Guang'an Vocational&Technical College,Guang'an,638000;Guang'an Science Park,Guang'an Vocational&Technical College,Guang'an,638000)

机构地区：[1]广安职业技术学院医学院,广安638000 [2]广安职业技术学院,广安科技园,广安638000

出　　处：《分子植物育种》2022年第1期225-231,共7页Molecular Plant Breeding

基　　金：干热河谷特色生物资源开发四川省重点实验室2019年开放基金项目(GR-2019-C-03)资助。

摘　　要：为了建立和完善旱半夏的ISSR-PCR反应体系,本研究以四川地区野生旱半夏为试验材料。以旱半夏整株的基因组DNA为模板,初筛的UBC818号引物用于ISSR-PCR体系,采用正交实验L_(16)(4^(5))并结合单因素实验方法对体系进行优化。通过正交试验结果直观评分可知各因素对该实验结果的影响强度从高到低依次为:d NTPs,引物,Mg^(2+),Taq DNA聚合酶,DNA模板;单因素实验结果表明,最优的20μL反应体系为:d NTPs 0.2 mmol/L,引物0.8μmol/L,Mg^(2+)2.0 mmol/L,Taq DNA聚合酶0.075 U/μL,DNA模板15 ng,退火温度为56℃。本研究通过优化ISSR-PCR反应体系,此体系将应用于半夏组培苗遗传稳定性的检测。In order to establish and perfect ISSR-PCR reaction system of Pinellia ternata,we used P.ternata from Sichuan region as the test material in this research.Genomic DNA of whole plant of P.ternata was used as tem plate,UBC818 primer was used in ISSR-PCR system,L_(16)(4^(5))orthogonal test and single factor experiment were used to optimize the system.Based on the visual analysis of orthogonal experiment,we found the following are the influences of various factors on the experimental results in turn:d NTPs,primer,Mg^(2+),Taq DNA polymerase,DNA template.Based on single factor screening test,we found the best ISSR-PCR reaction system.The total reaction volume is 20.0μL,containing d NTPs 0.2 mmol/L,primer 0.8μmol/L,Mg^(2+)2.0 mmol/L,Taq DNA polymerase0.075 U/μL,DNA 15 ng,annealing temperature 56℃.In this study,ISSR-PCR reaction system was optimized which will be applied to detect the genetic stability of P.ternata tissue culture seedlings.

关 键 词：旱半夏(Pinellia ternata) ISSR-PCR 优化 

分 类 号：S567.239[农业科学—中草药栽培]