热带作物学报2024，Vol.45Issue(4)：663-673,11.DOI:10.3969/j.issn.1000-2561.2024.04.002
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
摘要
Abstract
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 detec-tion 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 sin-gle 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 ex-ogenous 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 inte-gration 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PCRKey words
transgenic papaya/copy number/reference gene/digital PCR/fluorescence quantitative PCR分类
农业科技引用本文复制引用
谢秀菊,赵辉,夏启玉,刘帅,麦贤俊,贾瑞宗,郭安平,徐志胜,李峰,孔祥义..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[J].热带作物学报,2024,45(4):663-673,11.基金项目
三亚市科技创新专项(No.2022KJCX21) （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) （No.ZDKJ202002）
海南省院士创新平台资助项目. （）
