The Role Of RCD1 In ROS Homeostasis And Its Functional Analysis

Reactive Oxygen Species are produced in plants under normal growth and stress conditions,and a variety of signal transduction pathways in plants are activated to achieve redox balance through a variety of physiological reactions.RCD1(Radical-induced Cell Death1)is an important regulator of transcription factors involved in normal growth and development of plants,and plays an important role in many stress tolerance signaling pathways.Among them,RCD1 interacts with ROS-related retrograde signaling transcription factors ANAC013 and ANAC017 to maintain the redox balance between mitochondia and chloroplasts.Previous studies have shown that OCP3 and GPX3 may regulate plant growth and development through reactive oxygen pathway in Arabidopsis thaliana.In this research,bait and prey vectors were constructed,and the proteins OCP3 and GPX3 interacting with RCD1 were identified by yeast two-hybrid experiment.In order to identify the interaction sites,bioinformatic analysis was performed on RCD1.Based on the database results,primers were designed to obtain clones of different conserved domains of RCD1,and RST domain was finally confirmed as its interaction region.In addition,plant expression vectors of fusion protein tag sequences were constructed by homologous recombination method,and transgenic plants and hybrid lines were obtained,which provided experimental materials for the subsequent validation of protein interactions in vivo by immunoprecipitation.At the same time,the gene editing vectors of the interacting proteins OCP3 and GPX3 were constructed and the mutant materials were obtained to further understand the molecular mechanism of interaction between RCD1,OCP3 and GPX3 in regulating the ROS homeostasis in plants.The results of this study are as follows:1.pDEST32-RST?pDEST32-WWE?pDEST22-OCP and pDEST22-GPX were successfully constructed,and yeast two-hybrid experiment was performed to verify that RCD1 interacts with OCP3 and GPX3,and the interaction occurs through the RST domain.2.The CRISPR/Cas9 gene editing vectors pKEE401echerry-OCP3,pKEE401echerry-gpx3,pKEE401echerry-OCP3 & gpx3 with m Cherry visual screening function were constructed.Heterozygous mutant materials were obtained by screening.3.The plant expression vector containing the fusion protein tag sequence was successfully constructed by the recombinant technology.The increased gene expression was detected by the semi-quantitative PCR method and the phenotype of the transgenic plants was also observed.The transgenic plants were crossed to obtain the materials used for immunoprecipitation.