Study On The Regulatory Elements Of FAD2Gene And Its Trans-acting Factor In The Cultivated Peanut

Peanut (Arachis hypogaea L.) is one of the important oil crops, and the composition of fatty acid inseeds is an important criterion for evaluating the quality of peanuts. Peanuts or its related products, with ahigh ratio of oleic/linoleic acid （O/L）, possess better qualities and tolerant to oxidized corruption.Microsomal△12fatty acid desaturase (FAD2) catalyzes the conversion of oleic acid to linoleic acid, whichis the key enzyme in regulating the contents of oleic acid, linoleic acid and the O/L ratio in seed. Since theimportance of FAD2gene in the synthesis of unsaturated fatty acids in plants, it is meaningful to study themolecular mechanism on how it involves the synthesis and storage of lipids.Alternative splicing is a very important biological process in Eukaryotes, can make a gene to generatea plurality of matured mRNA transcripts, which enhances the plasticity of transcripts and the diversity ofproteins. The previous studies found that the alternative splicing occurs in the introns of the5’UTR regionof peanut AhFAD2A and AhFAD2B gene, which produced at least two different isoforms of spliceosomewith different expression modes in seeds. In this paper, to study the expression patterns of each alternativesplicing event of AhFAD2A, we investigated the regulatory mode of each promoter, respectively. In thisportion, the35S promoter of pBI121was replaced by different promoter fragments P1-P4of AhFAD2A,and the four expression vectors of GUS gene, named pBI121-AhFAD2A-P1, pBI121-AhFAD2A-P2,pBI121-AhFAD2A-P3and pBI121-AhFAD2A-P4were constructed and were transformed into Arabidopsis.Currently, transgenic Arabidopsis lines At-P1, At-P2, and At-P4with structures of AhFAD2A-P1,AhFAD2A-P2, and AhFAD2A-P4were obtained, and GUS histochemical staining was performed in roots,stems, leaves, flowers and seedlings of the positive transgenic plants. The results showed that the positivecontrol lines harboring pBI121have a strong expression of GUS in most detected tissues. The GUSstaining are positive in all detected tissues of transgenic Arabidopsis lines containing P4promoter, as wellas stronger expression of GUS gene occurs in stigma and anther of flower. GUS staining of other transgenicplants with other structures was in progress.Previous study by others found that the expression of SeFAD2in sesame seed was regulated bySebHLH, which interacts with E-box and G-box in the promoter of SeFAD2. In this study, we found that theupstream regulatory region of transcription start site in peanut AhFAD2A-2and AhFAD2B-2containsseveral E-box elements. Therefore, the1057bp fragment of AhbHLH cDNA was obtained through thestrategy of homologous cloning, which has an open reading frame with full-length of1050bp and encodes349amino acids. Multiple sequence alignment showed that there are only fewer differences in severalindividual amino acid residues between the conserved bHLH domain of AhbHLH and those of the knownbHLH transcription factors from other species. The results of yeast transcriptional activating experimentsshowed that AhbHLH had the function of transcriptional activation and the activating domain existed at theN-terminal. Subcellular localization displayed that the fused protein of AhbHLH-GFP was localized in thenucleus specifically. These results indicated that AhbHLH has the characteristics of transcription factor. The expression pattern of AhbHLH was analyzed by qRT-PCR, which showed that its expression displays theseed-specific pattern, and the highest level of expression occurs at the stage of40～50d after pegging,while there are almost no expression in roots, stems, leaves and flowers. Meanwhile, a plantover-expression vector, denominated pCAMBIA3301-M-AhbHLH, was constructed for functionalidentification of AhbHLH. Next, in order to explicit whether AhbHLH was involved in the regulation ofAhFAD2, the binding of AhbHLH with regulatory regions and candidate cis-acting elements of AhFAD2gene will be verified by electrophoretic mobility shift assay (EMSA).The analysis of AhFAD2promoters with different length, and the systematic researches oftranscription factor AhbHLH, will lay the foundation for clarifying the molecular mechanism of peanutAhFAD2involved lipid synthesis and storage, and will benefit for designing more reasonable strategy ofpeanut genetic improvement.