Functional Analysis Of OsCYP2 And Its Interactor OsZFP Via Controlling Lateral Root Development In Rice

Lateral roots(LR)are major components of rice root system.The development of LR is a postembryonic event.The morphological and physiological descriptions of LR development in plants have been extensively reported,however,little is known about molecular mechanisms of lateral root development in rice.Therefore,it is essential to identify the genetic determinants of lateral root development for breeding.In this study,we used the Oryza sativa L.cv.Aichi-ashahi(wild type,OsCYP2-overexpression and OsCYP2-RNAi)seedlings as the materials.Phenotype,RNA-seq and gene cloning were conducted to study the effect of OsCYP2 and its interacted protein(OsZFP)on LR formation in IAA signal pathway.The main results were as follows:1.OsCYP2 is a widely expressed gene in rice organs.The RNA interference(RNAi)of OsCYP2 resulted in the LR formation blocked and significant decrease of LR numbers.GUS staining results showed that grain,leaf,stem and root were stained blue.The color of grain was deepest,which revealed that the promoter of OsCYP2 was a constitutive promoter.After IAA treatment,it was observed that OsIAA1,OsIAA8 and OsIAA31 genes were dependent on OsCYP2 signal pathway.The expression level of Aux/IAA genes(OsIAA8,11,23 and 31)decreased significantly compared to WT in OsCYP2-RNAi lines,which showed insensitivity to auxin.2.RNA-seq results demonstrated that 3158 differentially expressed genes(DEGs)showed significant difference expression in OsCYP2 RNAi as compared to WT.Among these significant DEGs,1267 and 1891 genes were found as up-and down-regulated,respectively.Among them,11 significant DEGs were found to be involved in LR development,and another 4 DEGs were related to auxin.RT-PCR results showed that the increase/decrease tendency of above genes was coincidence with RNA-seq.Ribosome was the most enrichment pathway based on the analysis of significant DEGs in KEGG database,followed by flavonoid biosynthesis and biosynthesis of secondary metabolites.3.We constructed a cDNA library of O.sativa.Three positive interacted proteins were obtained,and they were amplified a single band by yeast colony PCR.In vitro GST-pull down proved that OsCYP2 interacted positively with OsZFP(0s01g0252900).Sequence and homology comparison revealed that OsZFP encoded a CCHC-type zinc finger protein and belonged to CCHC-type zinc finger proteins family.The phylogenetic analysis of OsZFP indicated that CCHC domain was highly conserved in Arabidopsis thaliana,Hordeum vulgare,Sorghum bicolor and Zea mays except in Homo sapiens,Saccharomyces cerevisiae and Triticum aestivum.4.Biological function of OsZFP,which was related to LR growth and development regulation,was studied using reverse genetic method.For detecting the function,the OsZFP gene was silenced in WT of O.sativa L.cv.Aichi-ashahi.pCAMBIA1300-OsZFP-RNAi vector was constructed and transformed into EHA105.Several OsZFP-RNAi independent lines were obtained.Phenotype of OsZFP-RNAi T2 seedlings demonstrated that the LR formation was blocked and the LR numbers were significantly decreased,which was the same as OsCYP2-RNAi.Subcellular location and co-location analysis showed that OsZFP was a nucleus located protein.Furthermore,after IAA treatment,RT-PCR data showed that the transcription level of six Avx/IAA genes(OsIAA1,2,6,20,23 and 24)was not significantly different.These results demonstrated that OsZFP-RNAi disrupted auxin-mediated signaling pathway required for LR formation.