An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts  

作　　者：Wenhui Yang Jiaqin Ren Wanrong Liu Dan Liu Kaidong Xie Fei Zhang Pengwei Wang Wenwu Guo Xiaomeng Wu 

机构地区：[1]National Key Laboratory for Germplasm Innovation&Utilization of Horticultural Crops,College of Horticulture and Forestry Sciences,Huazhong Agricultural University,Wuhan,Hubei 430070,China [2]Hubei Hongshan Laboratory,Wuhan,Hubei 430070,China

出　　处：《Horticultural Plant Journal》2023年第3期425-436,共12页园艺学报（英文版）

基　　金：supported by the National Natural Science Foundation of China;China (Grant Nos. 31872051, 32072528);the Foundation of Hubei Hongshan Laboratory (Grant No.2021hszd009)。

摘　　要：Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

关 键 词：CITRUS Callus protoplast Transient transfection Subcellular localization Genome editing 

分 类 号：S666[农业科学—果树学]