Cloning And Functional Analysis Of Peanut ERF Transcription Factor Genes During Plant Abiotic Stress Regulation

Abiotic stresses, including cold, drought and high salt, seriously affect the growth,development and yield of plants. A series of physiological and biochemical reactions are generated when plants are under abiotic stresses, and the expression of many stress-response genes are regulated during this adaptive process. Transcription factors can regulate the expression of downstream genes, and play important role in the regulation of gene expression related to abiotic stress. The transcription factors were known to be involved in the regulation of abiotic stress in plants include WRKY, NAC, AP2/ERF and MYB. The AP2/ ERF proteins have important functions in the transcriptional regulation of various responses to abiotic stress and the expression of many this family genes are induced by abiotic stress. Transgenic research shows that over-expression of many AP2/ERF genes can increase the resistance of plants to abiotic stresses.Peanut is an important oil crop nationwide and its growth and yield are seriously affected by abiotic stresses. However, there were limited studies conducted on abiotic stress regulation in peanut. In this article, six ERF family genes were screened and cloned from the peanut cDNA library. The sequence information and the expression pattern under abiotic stress were analyzed. Then the overexpression vector were constructed and transferred into Arabidopsis and peanut, respectively. The function and mechanism of these genes in the regulation of abiotic stress in plants were studied using transgenic plants.The following results were achieved through this study:1. Gene screening, cloning, sequence analysis and phylogenetic analysis(1) The total 40 ESTs,homologous with the AP2/ERF domain in the amino acid sequences of AtCBF1/DREB1B (UU77378) and AtCBF3/DREB1A (AF074602) of Arabidopsis, were searched from the peanut cDNA library constructed by ourselves.(2) Six AP2/ERF genes were cloned by common RT-PCR or via the rapid amplification of cDNA ends (RACE) method. Sequence analysis indicated that the amino acid sequences of the six proteins all contain a single AP2/ ERF conserved domain, which suggests they are ERF family proteins. We designated the genes as Arachis hypogaea ethylene-responsive element binding factor 1-6 (AhERF1,AhERF2, AhERF3, AhERF4, AhERF5, AhERF6).(3) An NCBI BLAST search indicated that these six ERF proteins have high similarity with the AP2/ ERF transcription factors in legume plants. The similarity in the sepuences of the AP2/ ERF domain is over 60 % with DREB/CBF proteins of Arabidopsis and legume plants.(4) A phylogenetic tree was constructed, based on the alignment of the AP2/ ERF domain of AhERFs with that of other ERF family proteins in Arabidopsis. The results indicated that AhERF2, AhERF3, AhERF5 and AhERF6 belong to the DREB/CBF subfamily (Group A)and AhERFl and AhERF4 belong to the ERF subfamily (Group B).2. Expression pattern analysis of AhERFs(1) Quantitative real-time PCR was employed to detect the expression patterns of AhERF1-6 in leaves, roots, stems, cotyledons, hypocotyls and flowers of peanut. The results reveal that all six ERF genes are expressed in the six peanut tissues investigated, but they have different expression levels and spatial patterns.(2) We monitored the expression changes of AhERF1-6 in peanut leaves and roots under abiotic stress. The results indicated that the expression of AhERF4 and AhERF6 were rapidly and substantially enhanced under abiotic stress. The expression of AhERF1 and AhEERF5 were slightly enhanced under certain stress conditions. Some genes were down-regulated when under stress, such as AhEERF3 in leaves under salt stress and AhERF2 in leaves under drought stress. Interestingly, the expression of AhERF3 and AhERF5 exhibited contrary expression patterns in peanut leaves and roots upon PEG treatment. These results suggested that different ERF proteins may have different functions in peanut abiotic stress acclimation.3. Function and mechanism analysis of AhERF6 in plant abiotic stress regulation(1) The protein characteristics of AhERF6 were studied. galactosidase activity experiment indicated that AhERF6 had transcriptional activity in yeast. Subcellular localization analysis showed that AhERF6 was located in the nucleus in plant cells. The two characteristics both indicated that AhERF is transcription factor.(2) The overexpression vector pCAMBIA1301-AhERF6 was transferred into Arabidopsis.The resistance of transgenic plants to low temperature and salt stress was significantly enhanced.(3) Mechanism analysis indicated the expression of stress-response genes RD29A, COR15A and KIN1 increased in transgenic plants compared with in wild-type, while there was no significant difference in, CBF3 expression. Over-expression of AhERF6 in Arabidopsis increased the resistance of transgenic plants to cold and salt stresses, possibly by activating the expression of some downstream stress-related genes in the DREB/CBF-signaling pathway.