青绿苔草CbSH4基因克隆及基因编辑靶点筛选OA

Carex breviculamis is an important native species that is water-saving and drought resistant,with advantages such as long green period,shade tolerance,and high fruiting rate.However,in the actual harvest-ing process,the problem of seeds easily falling off limits the mechanization of seed harvesting and large-scale production applications.In order to explore the function of SH4 gene in the process of seed-shattering in C.bre-viculmis and further use gene editing technology to create new germplasm resistant to seed shattering,the CbSH4 gene was cloned and the gene editing target was screened.The CbSH4 encoded 319 amino acid resi-dues,was a hydrophilic protein,and was most closely related to Carex littledalei,containing a highly con-served Myb4 DNA-binding domain,which belonged to a typical MYB transcription factor.The transient expression results of Nicotiana benthamiana showed that CbSH4 protein was localized in the nucleus and cyto-plasm.The real-time fluorescence quantification results showed that the relative expression level of CbSH4 in the spike was significantly higher than that in the roots and leaves,and it was expressed in the abscission zone at different developmental stages.At the same time,in order to achieve efficient gene editing in C.breviculmis,three specific targets were designed in this study,and sgRNA1 was obtained by sgRNA transcription and screening in vitro with a cleavage efficiency of 58.3%,which could be used for subsequent gene editing of C.breviculmis.In this study,CbSH4 was successfully cloned and the preliminary functional analysis was com-pleted,and the high-efficiency gene editing targets were screened,which provided a basis for the creation of new germplasm with low seed shattering.