Isolation And Functional Analysis Of Actin-depolymerizing Factor Gene ZmADF3 In Maize

Actin-depolymerizing factors(ADFs)are the members of actin-binding proteins,which play pivotal roles in various cellular processes in plant cells.In our previous study,differentially expressed protein libraries were constructed from pericarp and endosperm of two inbreds N04 and Dan232 at different developmental stages based on i TRAQ technology.Here we chosen one important protein peptide(GRMZM2G060702_P02)as our objective protein,for which its function was predicated as ADF3 and had a significant differentially expression between the two inbreds.In this study,ZmADF3 gene was isolated from the kernels of two inbreds,respectively.Quantitative real-time PCR and western blotting were performed to analyze the expression of ZmADF3 in the pericarp and endosperm at different developmental stages for the two inbreds during kernel development both at RNA and protein levels.Subcellular localization analysis was also done by the construction of the GFP fusion carrier for ZmADF3.Meanwhile,the promoter region for ZmADF3 was obtained and the cis-acting regulatory elements were predicted.Furthermore,yeast two-hybrid screening was carried out to screen the interacting proteins with ZmADF3.Finaly,heterologous overexpression of ZmADF3 in Arabidopsis to explore its functions.This study preliminarily revealed its functions in kernel development,which would be useful in further research to explore the function of ZmADF3 in maize kernel development.The main research results were as follows:1.A c DNA sequence fragment of ZmADF3 was obtained via PCR from inbred N04 as well as from the inbred Dan232,both of which contained an ORF of 417 bp encoding for 139 amino acids.Comparison of the two ORF sequences from N04 and Dan232 revealed that ten nucleotides were different,which resulted in three amino acid changes.Phylogenetic analysis showed that ZmADF3 had a high sequence identity with Si ADF3,Ta ADF3 and Os ADF4.2.Quantitative real-time PCR and western blotting analysis showed that ZmADF3 expressed abundantly at the early developmental stages of kernels,and the expression levels in RNA were much higher in pericarp than in endosperm at these stages.While,the expression tendency in protein levels in N04 also was much higher in pericarp than in endosperm,for which it was just opposite in Dan232.3.The GFP fusion vector p EGAD-EGFP-ZmADF3 was constructed and transformed into Arabidopsis,and the ZmADF3 protein was located in the cytoplasm by observing the seeding of the basta resistant T0 transformants using laser scanning confocal microscope.The promoter region of ZmADF3 was obtained from the genomic DNA of inbred N04.Then the cis-acting regulatory elements were predicted,in which many cis-acting regulatory elements associated with light,stress and hormone responses were found.4.The full-length ORF of ZmADF3 was cloned into the p GBKT7 vector and then transformed into the yeast strain Y2 HGold.After testing p GBKT7-ZmADF3 for autoactivation and toxicity,the c DNA library cloned inframe in the p GADT7 vector constructed from the kernels of inbred N04 was used to screen for the interaction clones with ZmADF3.There were 18 possible interacting proteins were detected and the co-transformation experiment confirmed that ZmADF3 could interact with GAPDH in yeast two-hybrid system.5.The overexpression vector p CAMBIA1302-ZmADF3 was constructed and transformed into Arabidopsis.There were a significantly higher expression in stem and inflorescence in homozygous progenies of transgenic lines.Compared with the WT plant,the 1000-seed weight of the transgenic lines increased significantly as well as the seed area,length and cotyledon areas of mature embryos resulted from the increase of the cotyledon cells.In addition,the positive regulation of seed size genes SHB1 and IKU1 were up-regulated while the negative regulators AP2 and ARF2 were down-regulated in the transgenic lines.Further study showed that the length of hypocotyl of ectopic expression seedlings increased 55.8% than that of the WT plant after 8 days darkness cultivation.The results suggested that ZmADF3 might increase seed size through promoting cell expansion and regulating the expression of seed size regulation genes in Arabidopsis,and light had an important influence on the regulation of ZmADF3 in plant growth and development.6.A digital gene expression(DGE)analysis was adopted to investigate the differences in gene expression between the 35S::ZmADF3 and wild type(WT).In total,26397 genes were detected.Among these genes,220 expressed significantly differentially and 160 were down-regulated and 60 were up-regulated in transgenic plants.GO analysis revealed that the differentially expressed genes involved in Chloroplast,Thylakoid and Photosystem in cellular component,Electron carrier activity and Binding activity in molecular function and Generation of precursor metabolites and energy,Fruit and seed development and Oxidation-reduction process in biological process.The pathway enrichment analysis of DEGs were performed and 63 pathway terms including Photosynthesis,Metabolic pathways,Phenylpropanoid biosynthesis,Starch and sucrose metabolism and Oxidative phosphorylation were enriched.