Subcellular Localization And Functional Analysisof DUF647 Family Member RUS4 From Arabidopsis Thaliana

ROOT UV-B SENSITIVE4(RUS4)is a member of the DUF647(Domain of Unknown Function 647,DUF647)in Arabidopsis thaliana.The function of RUS4 gene is unknown.In this study,ami R-rus4 plant was obtained by silencing RUS4 with artificial micro RNA technology.We used Arabidopsis ami R-rus4 plant as an experimental material to study the function of RUS4 gene through subcellular localization and gene expression analysis of auxin and jasmonic acid signaling pathway.Results are as follows :1.Ami R-rus4 plant is a male sterile lineCompared with WT plants,These lines exhibited normal vegetative growth,but showed reduced fertility.The ami R-rus4 plants produced very short siliques and a few seeds.RT-PCR and real-time quantitative PCR showed that RUS4 expression was down-regulated by about 40%.This suggests that the gene function of RUS4 may be related to fertility.2.RUS4 was located in chloroplastsIn order to better understand the function of RUS4,we performed its subcellular localization using a translational fusion to the Aequorea victoria green fluorescent protein GFP(RUS4-GFP).We first created the Entry clone using p ENTR directional TOPO cloning kit;subsequently,the RUS4 fragment in the entry clone was transferred to p MDC83 destination vector by using the Gateway LR Clonase enzyme mix.Then,we introduced the translational fusion 35Spro:RUS4-GFP into Arabidopsis(Col-0)plants.The distribution of RUS4-GFP was examined in mesophyll protoplasts.We found that the RUS4-GFP signal is highly localized to the chloroplasts,confirming that the protein is chloroplast localized.3.The gene expression of jasmonic acid signaling pathway changedThe male sterility in Arabidopsis thaliana was regulated by many factors,involving many signaling pathways.In jasmonic signaling pathway.Real-time fluorescence quantitative PCR was used to analyze theexpression of jasmonic acid signaling pathway genes.Among them,DAD1,OPR3 and AOS,which are key genes for jasmonic acid synthesis,were up-regulated in varying degrees.Jasmonate-ZIM domain protein(JAZ)JAZ7 gene expression was down-regulated.These changes suggest that RUS4 gene may regulate pollen maturation and anther dehiscence by affecting the Jasmonate-ZIM domain protein(JAZ)signaling pathway in plants.4.The gene expression of auxin signaling pathway changedIn auxin signaling pathway,YUC1 and YUC2,which encode auxin synthesis were up-regulated,indicating that auxin synthesis increased.At the same time,the expression of auxin transport vectors PIN5 and PIN8 also increased,and the expression of auxin-related genes affected the dynamic distribution of auxin.The staining of GUS driven by auxin response promoter DR5 showed that the distribution of auxin in root tips of DR5:ami R-rus4 plants was significantly expanded,and there was no significant difference in the distribution of auxin in root hairs,but auxin increased significantly in the shoot tips and leaf margins.These results suggest that RUS4 gene may regulate pollen maturation and anther dehiscence by affecting auxin signaling pathways in plants.In addition,RUS4 was also associated with changes in root hair density and root length.In conclusion,This study will facilitate further analysis of the function and molecular mechanisms by which RUS4 activity regulates Arabidopsis growth and development.