两种PCR快速检测转基因糜子中外源基因的插入拷贝数  

Rapid Detection of the Insertion Copy Number of Exogenous Genes in Transgenic Broomcorn Millet via Two PCR Methods

作　　者：董晓静 夏启玉[1] 降彦苗 刘国庆 程汝宏 赵辉[1] DONG Xiaojing;XIA Qiyu;XIANG Yanmiao;LIU Guoqing;CHENG Ruhong;ZHAO Hui(Sanya Research Institute,Chinese Academy of Tropical Agricultural Sciences/Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences/Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions,Sanya,Hainan 572024,China;College of Life Science,Luoyang Normal University,Luoyang,Henan 471934,China;Institute of Millet Crops,Hebei Academy of Agriculture and Forestry Sciences,Shijiazhuang,Hebei 050035,China)

机构地区：[1]中国热带农业科学院三亚研究院/中国热带农业科学院热带生物技术研究所/海南省南繁生物安全与分子育种重点实验室,海南三亚572024 [2]洛阳师范学院生命科学学院,河南洛阳471934 [3]河北农林科学院谷子研究所,河北石家庄050035

出　　处：《热带农业科学》2025年第2期55-63,共9页Chinese Journal of Tropical Agriculture

基　　金：三亚崖州湾科技城科技专项(No.SCKJ-JYRC-2022-90);中央引导地方科技发展资金项目(No.226Z6301G);国家自然科学基金(No.32241043)。

摘　　要：近年来,糜子(Panicum miliaceum L.)的转基因育种研究越来越多,本研究旨在建立简单快速检测转基因糜子中外源基因插入拷贝数的方法。从糜子基因组中筛选了一个单拷贝基因SD1,并以其为内参基因,以潮霉素抗性基因(Hyg)为外源基因,建立了数字PCR检测转基因糜子中外源基因插入拷贝数的方法。并以数字PCR鉴定的一个双拷贝样品为参照样本,建立了荧光定量PCR检测转基因糜子中外源基因插入拷贝数的方法。生物信息学分析表明,SD1为单拷贝基因,可作为转基因糜子中外源基因插入拷贝数检测的内参基因。数字PCR法检测的转基因糜子样品中,9份样品均为单拷贝或双拷贝插入;荧光定量PCR法检测的转基因糜子样品中,10份样品为单拷贝或双拷贝插入,其余13份样品均为多拷贝插入;对其中的4份低拷贝样品也进行了数字PCR检测,结果与荧光定量PCR法的检测结果完全一致,表明荧光定量PCR法检测转基因糜子中外源基因低拷贝时的准确度较高。本研究建立的数字PCR法简单快速,准确度高,适合在样品数量较少时使用,而荧光定量PCR法检测低拷贝插入时准确度较高,且成本低廉,适合从大量转基因株系中初筛出的低拷贝插入株系。这2种方法均可简单快速地检测糜子转基因育种中外源基因插入拷贝数。In recent years,there has been an increasing amount of research on the transgenic breeding of broomcorn millet(Panicum miliaceum L.).This study aimed to establish simple and rapid methods for detecting the insertion copy number of exogenous genes in transgenic broomcorn millet.This study screened the single-copy gene SD1 from the genome of broomcorn millet and established a digital PCR method to detect the insertion copy number of exogenous genes in transgenic broomcorn millet,using it as an internal reference gene and the hygromycin resistance gene(Hyg)as an exogenous gene.A fluorescence quantitative PCR method was established to detect the insertion copy number of exogenous genes in transgenic broomcorn millet,using a double copy sample identified by digital PCR as the reference sample.Bioinformatics analysis shows that SD1 is a single copy gene and can be used as an internal reference gene for insertion copy number detection of exogenous genes in transgenic broomcorn millet;Among the transgenic broomcorn millet samples detected by digital PCR,nine samples were either single copy or double copy insertions;Among the transgenic broomcorn millet samples detected by fluorescence quantitative PCR method,ten samples were single copy or double copy insertions,while the remaining 13 samples were multi-copy insertions.Among them,four low-copy number samples were also detected by digital PCR,and the detection results were completely consistent with those of the quantitative fluorescence PCR method,indicating that the quantitative fluorescence PCR method has high accuracy in detecting low-copy number insertions of exogenous genes in transgenic broomcorn millet.The digital PCR method established in this study is simple,fast,highly accurate,and suitable for use when the sample quantity is relatively small.On the other hand,the quantitative fluorescence PCR method has high accuracy in detecting low-copy insertions,and its cost is low,making it suitable for screening low-copy insertions from many transgenic strains.Both metho

关 键 词：转基因糜子 外源基因 插入拷贝数 数字PCR 荧光定量PCR 

分 类 号：S516[农业科学—作物学]