| Lotus corniculatus L.is used worldwide as a forage crop due to its abundance of secondary metabolites,high nutritive value and its ability to grow in severe environments.Members of the Apetala2/Ethylene Response Factor(AP2/ERF)family play important roles in regulating gene expression in response to many forms of stress,including drought and salt.1.In this paper,127 AP2/ERF genes were isolated from L.corniculatus var.japonicas by insilico cloning method.The phylogeny,gene structures,and putative conserved motifs in L.corniculatus var.japonicus EREBP proteins were analysed.Based on the number of AP2/ERF domains and the function of the genes,127 AP2/ERF genes from L.japonicus were classified into five subfamilies,named the AP2,DREB,ERF,RAV and a soloist,respectively.Outside the AP2/ERF domain,many L.japonicus-specific conserved motifs were also detected in these AP2/ERF genes.2.Results of the expression profile analysis of AP2/ERF genes by quantitative real-time PCR revealed that 19 LcERF genes were significantly induced by salt stress and 10 LcERF genes were significantly repressed by salt stress.Phenotype analysis of the 18 LcERFs-overexpressed-Arabidopsis under salt stress showed that the five LcERF genes LcERF053,LcERF054,LcERF056,LcERF080 and LcAP2/ERF107 were the salt stress-tolerant genes.3.RT-PCR strategy was used to analyze the expression of the five LcAP2/ERF genes LcERF053,LcERF054,LcERF056,LcERF080 and LcAP2/ERF107.Four of them were expressed in roots,stems,leaves,flowers and seeds,excluding the gene 2LcERF056 which was found to be expressed only in stems and leaves.In addition,qRT-PCR was performed to detect the expression patterns of the five LcAP2/ERF genes under different stress conditions,including ABA,ACC,JA,SA,and NaCl treatments.The results suggested that different signal pathways and models were involved in among the five genes.Results of subcellular localization analysis showed that all the five LcERFs were nuclear proteins.Results of yeast one-hybrid assays confirmed that four of the five TFs except LcERF080 protein have transcription activator.4.Overexpression of LcERF053,LcERF054,LcERF056,LcERF080 and LcAP2/ERF107 in Arabidopsis significantly enhanced the tolerances of transgenic plants to salt stress,showing that they are the important salt responsive LcAP2/ERF.Results of physiological and biochemical analysis indicated that the transgenic Arabidopsis showed higher germination ratio of seeds,elevated levels of relative water contents,soluble sugars,proline,and lower levels of alondialdehyde under stress conditions compared to wild-type plants.The expression of the major stress tolerance genes in the LcERFs-overexpressed-Arabidopsis were also investigated by qRT-PCR method.The results showed that the five genes LcERF053,LcERF054,LcERF056,LcERF080 and LcAP2/ERF107 enhanced the stress tolerance of transgenic Arabidopsis by regulating different stress tolerant genes of Arabidopsis.5.Results of yeast one-hybrid showed that LcERF056 protein binded to the GCC element,the transcriptional activation domain was located in 101-119 amino acids.6.Results of genetic transformation of L.corniculatus L.cv Leo showed that 20 transgenic plants were obtained,and LcERF056-transgenic Lotus corniculatus L.cv Leo resulted in enhanced tolerance to salt stress.These results revealed that the five genes LcERF053,LcERF054,LcERF056,LcERF080 and LcAP2/ERF107 play important roles in L.corniculatus L.cv Leo under salt stress.the LcERF genes are attractive engineering targets in applied efforts to improve abiotic stress tolerances in L.corniculatus L.cv Leo or other crops. |
PDF Full Text Request