Regulation Of Delta12Fatty Acid Desaturase Gene In Peanut

Peanut (Arachis hypogaea), as a major source of vegetable oil, is one of theimportant oilseed crops in the world. The main factor influencing oil quality of peanutseeds is the composition of fatty acids. Peanut oils with higher oleic acid contents areconsidered having higher nutritional value and longer shelf life. Microsomal delta12fatty acid desaturase (FAD2), a crucial enzyme involved in plant fatty acid synthesisand metabolism, catalyze producing linoleic acid through dehydrogenation of oleicacid at carbon12and introducing a double bond, which plays an important role onregulation the proportion of polyunsaturated fatty acids and saturated fatty acids in thestorage lipids and the membrane lipids of plant.In previous research, two copies of the AhFAD2gene (AhFAD2A and AhFAD2B)encoding the complete proteins were found in peanut genome, in addition, a kind ofpseudo-gene was existed. The discrepant contents of oleic acid and linoleic acid inpeanut seeds are highly deduced by the different activity of AhFAD2A and AhFAD2B,which associated with the sequence variation of the coding region of AhFAD2B andAhFAD2A. In order to study regulation of two genes, the upstream regulatory regionof AhFAD2A and AhFAD2B were amplified from LH14by the method of chromosome walking, and the following research was carried out.1. The transcription initiation sites of AhFAD2A and AhFAD2B were identifiedusing the method of5’RACE. It was found that the different four sites of two genesare located at the5′upstream sites of-99bp,-108bp,-112bp and-40bp fromtranslation start code ATG, respectively, suggesting that the intron of AhFAD2couldbe spliced in alternative sites. Due to intron alternative splicing, the distinct intronsand the corresponding promoters of these genes expressed as I1, I2, I3, I4and P1, P2,P3, P4were distingushed. Thus, the different transcripts of A1-A4or B1-B4maybeexist in different temporal and spatial pattern in peanut.2. Combining with the sequence analysis, the expression profiles of the differenttranscripts were preliminarily detected by the method of RT-PCR. The results showedthat the transcripts of A2, A4and B1, B2, B4were expressed in leaves, and thetranscripts of A2, A4in stems and roots, and in seeds, the transcript of A2, A3, A4andB1, B2were found. Furthermore, the expression patterns of AhFAD2A and AhFAD2Bgenes will be analyzed in different tissues and in the seeds with differentdevelopmental stage by quantitative real-time PCR.3. Bioinformatics analysis for the5′regulation region of AhFAD2A andAhFAD2B genes revealed that there were some cis-acting elements, such as RY, motif, E-box, G-box, ABRE motif, and etc., which control the expression of two genes in theseed. The upstream regulation sequences between AhFAD2A and AhFAD2B have64%identity. Among the rest, in the part of P4promoter, the sequence similarity is only21%, and17different nucleotides between the two intron regions of I4, which causethe difference of two cis-acting elements, share80%sequence similarity, suggestedmight to influence the expression pattern.4. Some introns or their alternative splicing involve in the regulation ofexpression level or temporal-spatial pattern of gene. In order to study the function ofintrons, the vectors harboring GUS gene driven by P4and the different intron I1, I2,I3, I4were constructed. Through transforming the gene into Arabidopsis thaliana, theexpression of the GUS gene was investigated. The results showed that GUS genedriven by P4and I4expresses in leaves, and in calyx and style of flower, and doesn’texpresse in root and petals of flower, and that some signals of GUS stain weredetectable in the stem. The study of other introns will be done nextly.In addition, another part of research has also been done. Previous studies showedthat it is an important method to improve the oleic acid content of oilseed crop in seedby inhibiting the expression of FAD2through gene engineering. As the targetsequence, the906-1120bp fragment in the ORF region of AhFAD2with many SiRNA target region, in the way of inverted repeat sequence, was inserted into the plant RNAivector with the p230promoter expressing in appropriate seed-specific mode. Aftertransferring this structure in peanut, we expect to inhibit or reduce the expression ofAhFAD2in seed specifically, and improve the oleic acid and linoleic acid contents ofseed.