| Maize (Zea mays L.) is one of the most important cereal crops used as the raw materials of grain,industrial and forage. Globally, maize ranks first in terms of planting area and total yield, exceed rice and wheat.Environmental factors that impose osmitic stress,such as salinity,drought and cold,place major limits on maize produetivity and quality. Water Stress is one of the most main abiotic stress factors that heavily influence the maize yield. Many genes responding water stress play important role in answering other stresses.Identification and function analyse of water-stress genes in maize are of great importance in increasing the water use efficiency, saving the water resources, reducing the damage caused by drought, and achieving sustainable development in the agriculture.The ERD genes are a member of the genes expressed in response to dehydration-related environment stresses suddenly. ERD genes are indentified firstly in Arabidopsis after one-hour dehydration,which includes 16 members,such as AtERD16. AtERD16,as a ubiquitin extension protein,protect plant cell by degradate exceptional protein. We describe here the isolation ZmERD16 gene encoding a homologue of ubiquitin extension protein wich AtERD16 from the drought-tolerant maize inbred line CN165. The genetie struetures were predieted by bioinformatics tools,and evolutionary analysis of ZmERD16 gene were performed. The expression of ZmERD16 gene was identificated by real-time PCR .The gene was transferred into Arabidopsis to study gene fuction prediction.The main results were as follows:1. Cloning ZmERD16 gene by homology cloningA ubiquitin extension protein gene was cloned in drought-tolerant maize inde line CN165 by homology cloning method.The similarity of the fragment and AtERD16 gene sequence is quietly high,named as ZmERD16. While the genomic DNA of ZmERD16 with 3252 bp and the promoter region with 2190 bp was obtained by PCR method2. Sequence analysis of ZmERD16 geneZmERD16 included a 390 bp open reading frame (ORF) encoding an ubiquitin monomer followed by 53 animo acids, with a predicted molecular mass of 14.7582 kD and PI of 9.94. The genomic DNA was composed of 4 exons and 3 introns.3. Homology comparison and phylogenetic analysisA homology search against the GenBank database showed that ZmERD16 is homologous to various ubiquitin/L40 proteins in eukaryotes.To investigate the evolutionary relationships among different ubiquitin/L40 proteins in plant organisms,a phylogenetic tree was constructed using diverse plant organisms against the GenBank database.The tree could clearly divide into three big groups:monocots,eudicots and green algae.4. Subcellular location of ZmERD16Subcellular localization analysis in onion epidermal cells showed that ZmERD16 expressed transiently in cytoplasm and nucleus.5. Promoter motif search of ZmERD16Promoter had some motifs that were related to light, stress, defense, development, auxin and others stresses.6. Expression analysis of ZmERD16 gene in maizeThe tissue-specific expression analysis suggested that certain ZmERD16 expression level were detected in shoots, roots, seeding roots, female ear, tassels, immature spikes, silk, ear leaves of maize under normal growth conditions. Qutitive real-time RT-PCR results showed ZmERD16 was a multiple stresses inducible gene, induced by various stresses, such as salt, dehydration, cold, heat, PEG, MJ and SA, but not induced by ABA and 2,4-D.7. Over-expression of ZmERD16 in ArabidopsisIn early stages of vegetative growth, rosette leaves of 35S:ZmERD16 overexpression plants were smaller , however, flowering delayed than those of wild-type plants development of Arabidopsis ;The seeds germination rate of ZmERD16 overexpression lines in NaCl-MS medium and livability of ZmERD16 overexpression lines in drought or NaCl stress condition were higher than the control wild-type. |
