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

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。

Abstract

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

Key words

Melon / CRISPR/Cas9 system / Agrobacterium rhizogenes / Gene knockout

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基金资助

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)