Cloning And Characterization Of AP2/EREBP Family Transcription Factor Genes From Elytrigia Elongata

Abiotic stresses, e.g. drought,salt, and low temperature, could have profound effects on growth and development of plants, which are also the direct reasons of yield loss of crops. The environmental stress factors could induce a series of physiological and biochemical changes in plants, from which one kind of proteins called transcription factors can specifically bind the cis-elements in the promoter region of eukaryotic genes, and regulate transcriptions of a series of downstream genes through their interactions with cis-elements or related proteins. Plant AP2/EREBP family proteins could activate the expression of a series of stress resistant genes in response to adverse enviromental factors such as drought, high salt and low temperature, which make them more significant in the field of research and application for improving the comprehensive stress resistances of crops.In this study, we obtained two AP2/EREBP family full-length cDNAs from Elytrigia elongata (2n=70) through homologous cloning method by a combined techniques of RACE and Genome Walking. The two cDNAs were named EeAP2-1.1 and EeAP2.2 respectively. Sequence analysis indicated that EeAP2-1.1 contains an ORF of 1179 bp, which encodes a protein of 392 amino acids, and the typical conserved AP2 domain in the amino sequences classified it as a new member of AP2 gene family. Further comparison analysis through NCBI blast showed that it is a ERF transcription factor belonging to EREBP subfamily. It has a 77% amino sequence identity with HvERF1 (GenBank accession No.: ADO21119.1) from Hordeum vulgare, and 76%, 76% and 73% amino sequence identities with genes whose GenBank accession No. are EAZ09217.1, AAV98700.1 and AAM00285.1 from Oryza sativa, respectively, and a 76% amino sequence identity with a gene whose GenBank accession No. is ABO09809.1 from Saccharum arundinaceum. EeAP2.2 contains an ORF of 837 bp, which encodes a protein of 278 amino acids. It also contains a conserved AP2 domain in the amino sequences which indicate it is a new member of AP2 gene family. It has 98% amino sequence identities with TaDREB1 (GenBank accession No.: AAL01124.1) and TaDREBW50 (GenBank accession No.: AAY44605.1) from Triticum aestivum, and its amino sequence identities with HvDREB1-a (Accession No.: AAY25517.1) from Hordeum vulgare, FaDREB2A (Accession No.: CAG30547.1) from Festuca arundinacea and OsDREB2.2 (Accession No.: AY064403) from Oryza sativa are 93%, 86%, 69%, respectively. The results showed that EeAP2.2 has high amino sequences identities with homologous from Triticum aestivum.Meanwhile, the eukaryotic expression vector pBI121- EeAP2.2 and pBI121- EeAP2-1.1 containing CaMV 35S promoter were constructed and transformed into tobacoo by Agrobacterium-mediated leaf-disk method. Presently, we have obtained the EeAP2.2 transgenic tobacoos. Using tobacco genome DNAs as PCR templetes, we have proved that EeAP2.2 have been integrated into the genome of tobacco transgenic lines. Using GUS histochemical staining method, we proved one transgenic line have successfully expressed the GUS fused EeAP2.2 proteins. In addition, the yeast one-hybrid method showed that EeAP2.2 can specifically bind to DRE cis-element, thus EeAP2.2 could be classified as DREB-like protein. The experiments for characterizing the resistance mechanism and functions of EeAP2.2 and EeAP2-1.1 and their transformation in wheat cultivars are in progress .Taken together, this study not only obtained two important AP2/EREBP family transcription factor genes from Elytrigia elongata, but also provided a fast and effective method for cloning full length cDNA sequences in plants. The results of this study also laid a basis for further clarifying the roles of AP2/EREBP family genes in plant growth, development and stress resistance, etc.