Function Analysis Of Small G Protein Gene ZmArf2 On Growth And Development

Small GTP-binding proteins are G proteins with a molecular weight between 20 and 30 k Da and play an important role in signal transduction and physiological processes in many plants.In our previous study,the differentially expressed genes of early endosperm development for a normal maize inbred line Dan232 with large grain size and a popcorn inbred line N04 with small grain size were isolated by suppression subtractive hybridization,and the small G protein gene ZmArf2 was cloned and transformed into Arabidopsis.We found that ZmArf2 could increase organ and seed size by promoting cell expansion in Arabidopsis.In order to further study the effect of ZmArf2 on plant growth and development,the interaction proteins with ZmArf2 protein were verified by yeast two-hybrid rotation assay and GST Pull-down in vitro,and overexpressed ZmArf2 transgenic Arabidopsis and wild type were study for digital gene expression profiling.This study confirmed the proteins interacting with ZmArf2 and analyzed the changes in the physiological processes of Arabidopsis from the expression level.The main results were as follows:1.Digital gene expression profiling was performed on transgenic and wild-type plants grown for 5 weeks.A total of 26597 genes were identified,and 745 genes were significantly differently expressed,of which 521 were down-regulated and 224 were up-regulated.Eight randomly selected DEGs were performed real-time PCR,and the result showed that the expression profile data was reliable.The most prominent components of the GO classification were the chloroplasts,thylakoids,and photosynthetic systems.The molecular functions mainly included oxidoreductase activity,electron carrier activity and tetrapyrrole binding.The biological processes involved metabolites,synthesis and energy transfer.These enriched GO functional classifications were closely related to plant growth and development.We obtained 12 differentially expressed genes involved in fruit and seed development,which indicated that ZmArf2 affected the growth and development of Arabidopsis.There are 507 differentially expressed genes(DEGs)annotated by Pathway analysis.These DEGs involved a total of 105 Pathways.The most prominent top 5 entries were photosynthesis,metabolic pathway,oxidative phosphorylation,secondary metabolites,biosynthesis and ribosome formation.Several DEGs annotations were related to ABA response.ABA hormone treatment experiments showed that ZmArf2 could resist ABA stress to a certain extent.2.The yeast expression vector pGBKT7-ZmArf2 and 4 yeast expression vectors pGADT7-Zm ISP1 、 pGADT7-Zm MT2 A 、 pGADT7-Zmvps29 and pGADT7-ZmSYS1 of the interaction genes were constructed.The bait protein and the interacting protein were co-transformed into yeast cells by yeast double-hybrid rotation verification.As a result,the cells grew normally on the two-deficient medium,and grew normally on the four-deficient+X+A medium and the colonies turned blue.It was demonstrated that ZmArf2 protein interacted with Zm ISP1、Zm MT2A、Zmvps29 and ZmSYS1 proteins in the yeast double-hybrid system.3.GST-tagged protein expression vector pGEX4T-1-ZmArf2 and His-tagged protein expression vector p ET28a-Zm ISP1、p ET28a-Zmvps29 and p ET28a-ZmSYS1 were constructed,and transformed into E.coli BL21 competent.IPTG was added for induction,these proteins can be expressed in prokaryotic systems at a certain temperature and time.Furthermore,it was found that there was a real interaction between ZmArf2 protein and Zm ISP1、Zmvps29 和 ZmSYS1 proteins by GST Pull-down and western-blot analysis.4.In this study,an interesting interaction protein ZmSYS1 was selected for Arabidopsis transformation.Transgenic ZmSYS1 gene Arabidopsis homozygous strains and Columbia wild-type Arabidopsis were used for transformation.The overexpression vector p CMBIA3301-ZmSYS1 was constructed and transformed into agrobacterium tumefaciens GV3101,which were transformed into wild-type and transgenic ZmArf2 gene Arabidopsis by pollen tube channel method to obtain positive plants.The preliminary phenotypic identification found that the ZmArf2 gene and the interaction gene ZmSYS1 affected the flowering period of Arabidopsis,and ZmSYS1 could enhance the expression of ZmArf2 in the roots of Arabidopsis.