数字PCR和荧光定量PCR检测转基因番木瓜中外源基因拷贝数方法的建立及其应用  

Establishment and Application of Digital PCR and Fluorescence Quantitative PCR for Detection of the Copy Numbers of Exogenous Gene in Transgenic Papaya

作　　者：谢秀菊 夏启玉[1] 刘帅 麦贤俊 贾瑞宗[1] 郭安平[1] 徐志胜[2] 李峰 孔祥义 赵辉[1] XIE Xiuju;XIA Qiyu;LIU Shuai;MAI Xianjun;JIA Ruizong;GUO Anping;XU Zhisheng;LI Feng;KONG Xiangyi;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;Nanjing Agricultural University,Jiangsu,Nanjing 210095,China;Sanya Academy of Tropical Agriculture,Sanya,Hainan 572022,China;Bellagen Biotechnology Co.,Ltd.,Jinan,Shandong 250300,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]南京农业大学,江苏南京210095 [3]三亚市热带农业科学院,海南三亚572022 [4]山东舜丰生物科技有限公司,山东济南250300

出　　处：《热带作物学报》2024年第4期663-673,共11页Chinese Journal of Tropical Crops

基　　金：三亚市科技创新专项(No.2022KJCX21);海南省重大科技计划项目“南繁育种区生物安全防控”(No.ZDKJ202002);海南省院士创新平台资助项目。

摘　　要：传统的检测转基因植物中外源基因拷贝数的方法是Southern杂交,该方法成本高、周期长,难以满足高通量检测外源基因拷贝数的育种需求,因此,本研究旨在建立快速且高通量检测转基因番木瓜中外源基因拷贝数的方法。本研究从番木瓜基因组中筛选出2个单拷贝基因Cpa03g018830和Cpa03g018770,以已知外源基因为单拷贝整合的转基因番木瓜为参照,利用数字PCR鉴定其拷贝数,进一步以其为内参基因,以番木瓜转基因育种常用的筛选标记基因NPTⅡ为外源目的基因,建立利用数字PCR和荧光定量PCR技术检测转基因番木瓜中外源基因拷贝数的方法。结果表明:Cpa03g018830和Cpa03g018770均为单拷贝基因;建立的数字PCR方法对转基因番木瓜中的外源基因拷贝数的检测准确可靠,而荧光定量PCR对转基因番木瓜中外源基因单低拷贝整合的检测准确度较高,对外源基因多拷贝整合的检测准确度则较低,因此,荧光定量PCR适合用来在大量转基因植株中初筛出单低拷贝整合的植株。本研究鉴定的单拷贝基因Cpa03g018830和Cpa03g018770可作为转基因番木瓜中外源基因拷贝数检测的内参基因,且建立的利用数字PCR和荧光定量PCR技术检测转基因番木瓜中外源基因拷贝数的方法,操作简单,速度快,适合批量检测,可为番木瓜转基因抗病育种中单低拷贝株系的选育提供新的方法。The traditional method for detecting the copy numbers of exogenous gene in transgenic plants is Southern hybridization,which is costly and time-consuming,and is difficult to meet the breeding needs of high-throughput detection of the copy numbers of exogenous gene.Therefore,this study aims to establish a fast and high-throughput method for detecting the copy numbers of exogenous gene in transgenic papayas.In this study,two single copy genes,Cpa03g018830 and Cpa03g018770,were selected from the papaya genome.Using the known exogenous gene as a single copy integrated transgenic papaya as a reference,the copy numbers were identified by digital PCR.Further,using them as reference genes,and the commonly used screening marker gene NPTII in papaya transgenic breeding as an exogenous target gene,methods for detecting the copy numbers of exogenous gene in transgenic papaya using digital PCR and fluorescence quantitative PCR were established.The results showed that Cpa03g018830 and Cpa03g018770 were both single copy genes;the established digital PCR method was accurate and reliable in detecting the copy numbers of exogenous gene in transgenic papayas,while fluorescence quantitative PCR had a higher accuracy in detecting single or low copy integration of exogenous genes in transgenic papayas,and a lower accuracy in detecting multiple copy integration of exogenous genes.Therefore,fluorescence quantitative PCR is suitable for initially screening out single or low copy integration plants from a large number of transgenic plants.The single copy Cpa03g018830 and Cpa03g018770 identified in this study can be used as reference genes for detecting the copy numbers of exogenous gene in transgenic papayas.The established methods for detecting the copy numbers of exogenous gene in transgenic papayas using digital PCR and fluorescence quantitative PCR techniques are simple,fast and suitable for batch detection,providing new methods for selecting single or low copy lines in transgenic resistance breeding of papayas.

关 键 词：转基因番木瓜 拷贝数 内参基因 数字PCR 荧光定量PCR 

分 类 号：S667.9[农业科学—果树学]