发根农杆菌介导的甜瓜CRISPR/Cas9系统靶位点的检测  

Detection of target sites of CRISPR/Cas9 system in melon by Agrobacterium rhizogenes system

作　　者：朱蕾[1] ZHU Lei(Daqing Branch of Heilongjiang Academy of Agricultural Sciences,Daqing 163711,Heilongjiang,China)

机构地区：[1]黑龙江省农业科学院大庆分院,黑龙江大庆163711

出　　处：《中国瓜菜》2024年第8期15-23,共9页China Cucurbits And Vegetables

基　　金：黑龙江省农业科技创新跨越工程优青项目(CX22YQ32);黑龙江省农业科技创新跨越工程农业特色产业项目(CX23TS11)。

摘　　要：选取甜瓜栽培材料龙庆八号作为受体材料,构建CmCURT1A基因CRISPR/Cas9基因编辑载体,经发根农杆菌介导检测靶位点的编辑情况,为后续甜瓜遗传转化试验提供载体基础。以甜瓜CmCURT1A基因(ID:MELO3C006053.2)为靶基因构建双靶位点敲除载体,经发根农杆菌K599介导的简单遗传转化技术使甜瓜组织长出不定根,经PCR测序发现在不定根中分别存在65 bp、72 bp不同碱基片段的缺失。该方法成功进行了甜瓜CRISPR/Cas9载体靶位点敲除情况的检测,简单高效,实现了在甜瓜中基因编辑靶点的快速鉴定,为研究甜瓜基因功能和遗传改良奠定基础。The cultivation melon Long Qing No.8 was used as the receptor material to construct the CRISPR/Cas9 editing vector of CmCURT1A gene.The targe site was detected through Agrobacterium rhizogenes,which provided the vector basis for subsequent genetic transformation experiment of melon.A double target knockout vector was constructed using the CmCURT1A gene(ID:MELO3C006053.2)as the target gene.Through a simple genetic transformation technique mediated by Agrobacterium rhizome K599,adventitious roots were grown.PCR sequencing revealed that different base fragments of 65 bp and 72 bp were absent in the adventitious roots.The method was simple and efficient for the detection of CRISPR/Cas9 vector target site knockout in melon,realizing rapid identification of gene editing targets,and laying a foundation for studying the gene function and genetic improvement in melon.

关 键 词：甜瓜 CRISPR/Cas9 发根农杆菌 基因敲除 

分 类 号：S652[农业科学—果树学]