Isolation And Functional Analysis Of Ethylene-signaling Pathway Genes ZmERF1 And ZmEIL1 In Maize (Zea Mays L.)

Maize(Zea mays L.),one of major crops,plays an important role in the food security in China.At present our country faces a growing population and reducing cultivated land area continuously,it is an effective way to ease the food security problem by increasing maize yield and quality.Biotic and abiotic stresses,especially heat,salinity,and osmotic stress,have adverse effects on maize production.Transcription factors play key roles in regulating plant stress responses through binding to cis-acting elements in the promoters of stress-related genes or interacting with other transcription factors,subsequently activating or repressing the expression of related genes.The AP2/EREBP family,a unique gene family in plants,plays important roles in plant growth,development,metabolism and defense response.However,the function of the ERF subfamily has been less known in maize.Here,we isolated two transcription factors involved in ethylene signal transduction pathway in maize,ZmERF1 and ZmEIL1.Their expression patterns in maize endosperm development,responses to abiotic stresses,subcellular localizations,and GCC box-binding activities were analyzed.The function of ZmERF1 in Arabidopsis was also studied.These results uncover the function of ZmERF1 and ZmEIL1 in the kernel development and abiotic stresses,which will lay good foundation for mining excellent germplasm resources and reveal ethylene signal transduction pathway in maize.The main results were as follows:1.A cDNA sequence,named ZmERF1,was amplified and cloned via RT-PCR from inbreds N04 and Dan232,respectively.The ORFs include 687 bp in inbred N04 and 690 bp in Dan232,whitch encoded 229 and 230 amino acids,respectively.There are 5 insertions,deletions and substitutions between the two ORFs.The ZmERF1 protein contains a single conserved AP2 DNA-binding domain and a bipartite nuclear localization signal(NLS).The ZmERF1 protein is much highly homologous with sorghum Sb ERF and rice Os ERF.The ZmERF1 protein was located in the nucleus by transforming the ZmERF1::GFP construct into onion epidermal cells.The ZmERF1 protein could function as a transcriptional activation and bind with GCC-box cis-element in yeast cells.These showed that ZmERF1,an ethylene responsive transcription factor,was classified as ERF subfamily of the AP2/EREBP family.2.The expression pattern of ZmERF1 in the developing endosperm throughout kernel development showed that ZmERF1 expressed much highly in mid-stage and later-stage of endosperm development.It was the same expression pattern for the ZmERF1 protein by Western Blot.Ethylene releasing rate was also high value in mid-stage and later-stage of endosperm development.This suggested that ZmERF1 expression could be involved in grain-filling and dehydration in maize.3.The cis-elements including ABA-responsive elements,ethylene-responsive elements,abiotic stress elements were detected in ZmERF1 promoter region.The expression level of ZmERF1 could be induced by Na Cl,heat,PEG,ABA and ethylene in maize seedlings at V4 stage.The vector of ZmERF1 promoter::GUS was constructed and transformed into Arabidopsis.The result showed that the expression level of GUS was much high in young leaves and inflorescence.Furthermore,GUS activity was strikingly stronger in the flower by salt and osmotic stress.These results indicated that ZmERF1 could be involved in the responding to abiotic stress.4.The over-expression vector of ZmERF1 was constructed and transformed into Arabidopsis.The results showed that only 3% of WT plants survived after recovery from heat stress,whereas 53% in the transgenic plants(OE15).After PEG treatment,the proline content in the transgenic plants was 2 times of WT.Under high salt stress,the chlorophyll content of transgenic plants with 2.193 mg/g was significantly higher than that of WT with 1.625 mg/g.These showed that ZmERF1 could improve the resistance ability to high temperature,penetration and salt stresses in Arabidopsis.Under normal growth conditions,the expression level for stress resistance genes(KIN2 and RD29A),ethylene biosynthesis genes(ACS2,ACS5)and ABA biosynthetic genes(NCED5,SDR)of transgenic plants were significantly higher than that of WT.These showed that the over-expression of ZmERF1 could efficiently activate expression level of downstream stress tolerance genes,ethylene biosynthesis genes and ABA biosynthetic genes,which could improve adversity stress resistance by the ABA and ethylene signal transduction pathways.5.One gene named ZmEIL1 involving ethylene signal transduction pathway in maize were amplified and cloned via RT-PCR from inbreds N04 and Dan232.The ORFs were 1941 bp and 1944 bp,with encoding 647 and 648 amino acids in inbreds N04 and Dan232,respectively.The ZmEIL1 protein contains 5 BD regions,5 ?-helices and a nuclear localization signal(NLS)(GPPPYKKPHDLKKAW).The ZmEIL1 protein was located in the nucleus.The ZmEIL1 protein showed highly homologous with rice Os EIL.The expression of ZmEIL1 could be induced by ethephon in maize leaves and roots.But it was repressed by 1-MCP in maize leaves,but not in root.These showed that the expression levels of ZmEIL1 were regulated by exogenous ethylene.In the transgenic plants of over-expression ZmERF1,both At EIN3 and At EIL1 showed constitutive high expression level in Arabidopsis.These suggested that ZmEIL1 was an important transcription factor involved in ethylene signal transduction pathways.