发根农杆菌介导的甜瓜转化体系的建立及对CRISPR/Cas9靶位点的快速检测  

Establishment of Agrobacterium rhizogenes-mediated Transformation System in Melon(Cucumis melo)and Rapid Detection of CRISPR/Cas9 Target Sites

作　　者：舒铫 杨嵩涵 孔可心 吕若涵 吕桂云[3] 张春秋[2] 宋士清[1] SHU Yao;YANG Song-Han;KONG Ke-Xing;LYU Ruo-Han;LYU Gui-Yun;ZHANG Chun-Qiu;SONG Shi-Qing(College of Horticultural Science&Technology,Hebei Normal University of Science&Technology/Hebei Key Laboratory of Horticultural Germplasm Excavation and Innovative Utilization/Hebei Higher Institute Application Technology Research and Development Center of Horticultural Plant Biological Breed,Qinhuangdao 066004,China;Beijing Vegetables Research Center,Beijing Academy of Agriculture and Forestry Sciences/State Key Laboratory of Vegetable Biobreeding/National Engineering Research Center for Vegetables/Beijing Key Laboratory of Vegetable Germplasms Improvement/Key Laboratory of Biology and Genetics Improvement of Horticultural Crops(North China)/Key Laboratory of Urban Agriculture(North)of Ministry of Agriculture and Rural Affairs,Beijing 100097,China;College of Horticulture,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北科技师范学院园艺科技学院/河北省特色园艺种质挖掘与创新利用重点实验室/河北省高校特色园艺植物生物育种应用技术研发中心,秦皇岛066004 [2]北京市农林科学院蔬菜研究所/蔬菜生物育种全国重点实验室/国家工程技术研究中心/蔬菜种质改良北京市重点实验室/华北地区园艺作物生物学与种质创制重点实验室/农业农村部华北都市农业重点实验室,北京100097 [3]河北农业大学园艺学院,保定071000

出　　处：《农业生物技术学报》2025年第4期924-932,共9页Journal of Agricultural Biotechnology

基　　金：北京市农林科学院创新能力建设专项科技攻关项目(西甜瓜等特色蔬菜关键品质性状分子基础解析及高品质品种选育);北京市农林科学院青年基金项目(QNJJ202205);北京市农林科学院创新能力建设项目(KJCX20230221);北京市农林科学院蔬菜研究所改革与发展项目(KYCX202301);国家自然科学基金面上项目(32072602)。

摘　　要：利用CRISPR/Cas9系统进行基因编辑是研究甜瓜(Cucumis melo)基因功能和进行遗传改良的有效方法,但目前缺少在遗传转化前能够快速检测CRISPR/Cas9载体靶位点的方法。本研究以甜瓜八氢番茄红素脱氢酶(phytoene desaturase,PDS)基因CmPDS为靶标,构建CRISPR/Cas9系统双靶点的基因敲除载体,通过发根农杆菌(Agrobacterium rhizogenes)K599侵染厚皮甜瓜'K7-2'和薄皮甜瓜'LB'的子叶外植体,利用Bar试纸条对诱导出的毛状根进行鉴定,并对毛状根进行测序分析以明确基因编辑类型。结果显示靶点1的编辑效率为100%,靶点2的编辑效率为76.5%,存在多种编辑类型,包括单碱基和多碱基的插入、缺失,以及2靶点间大片段缺失。本研究成功建立了发根农杆菌介导的甜瓜遗传转化体系,实现了对CRISPR/Cas9载体靶位点的快速检测,验证了CRISPR/Cas9在甜瓜基因编辑中的有效性,为甜瓜基因功能研究和遗传改良提供了技术支持。The utilization of the CRISPR/Cas9 system for gene editing is an effective method for investigating gene functions and pursuing genetic improvement in melons(Cucumis melo).However,there lacks a method that rapidly detects the target sites of CRISPR/Cas9 vectors prior to genetic transformation.In this study,the melon phytoene desaturase(PDS)gene CmPDS was used as target gene,two sgRNAs were designed and introduced together into the CRISPR/Cas9 vector.Agrobacterium rhizogenes K599 was used to infect cotyledon explants of the thick-skinned melon'K7-2'and the thin-skinned melon'LB'.The induced adventitious roots were firstly identified using Bar test strips,and then the target regions were sequenced to see if the regions were edited and clarified the types of mutation.The PCR products sequencing results showed that the 2 target sites were all edited.Further sequencing of the individual PCR products demonstrated an editing efficiency of 100%for target 1 and 76.5%for target 2,with multiple mutation types observed,including insertions and deletions of single or multiple bases,as well as large deletions between 2 target sites.This study successfully established a genetic transformation system for melons that was mediated by the A.rhizogenes,and achieved rapid detection target sites of CRISPR/Cas9 vector,and also validated the effectiveness of using the CRISPR/Cas9 system in melon gene editing.This study provides an important technical support for genetic functional studies and genetic improvements of melons.

关 键 词：甜瓜 CRISPR/Cas9 发根农杆菌 基因编辑 

分 类 号：S652[农业科学—果树学]