Molecular Characterization Analysis Of Oil Synthesis Key Enzyme Genes DGAT3and SAD And Genetic Transformation Of FAD2in Peanut

Cultivated peanut （Arachis hypogaea L.） is a widely cultivated commercial plant in theworld，which is also an important oilseed and cash crop in China. Triacylglycerols （TAG） arethe main storage lipids in most oil plants. A major determinant of oil quality of peanut is thecomposition of fatty acids. Peanut oils with higher content of oleic acid are favorable forhuman being because of their improved oil stability and enhanced nutritional quality.Diacylglycerol acyltransferase （DGAT） is the rate-limiting enzyme that catalyzes the finalstep in TAG biosynthesis by converting diacylgycerol （DAG） and fatty acyl-coenzyme A（CoA） into triacylglycero1. The key enzymes, delta9stearoyl–acyl carrier protein （ACP）desaturase （SAD） and delta12fatty acid desaturase （FAD2）, involve in regulation of oleicacid content. Based on PCR, we obtained DGAT3DNA sequence in peanut for the first time.Two full-length DNAs of DGAT3were cloned from each of twelve varieties of A. hypogaeaand one was cloned from each of six wild species. By comparing the sequence homology, westudied the genetic relationship between cultivated peanut and each wild species involved. Byusing the primers based on the peanut cDNA sequence of SAD （AF172728）, genomic DNAswere amplified from the wild diploid species A. duranensis and A. ipaensis, and from3cultivated peanut accessions. In addition, two isoforms of SAD cDNA were isolated and theexpression vector of pSAD-2was constructed. What’s more, the antisense ahFAD2B wasintroduced into peanut cultivar and T0and T1positive transgenic seedlings were obtained. Themajor results are abstracted as follows:（1） Cloning and sequence analysis of DGAT3in peanutTwo isoforms of DGAT3cDNA were isolated and named as ShrDGAT3-1andShrDGAT3-2, which share96.9%identity in nucleotide level. Deduced amino acid sequencesrevealed that there were ten amino acid variations. They were identified potential DGATmotifs and partially conserved DGAT active site, which matched with reported members ofthe acyltransferase family. But thiolase acyl enzyme intermediate signature displayed fouramino acid deletions between residues¹³⁵TNPDCESSSSSSSSESESES¹⁵⁴.Two isoforms of thegenomic DGAT3were identified and designated as gDGAT3-1and gDGAT3-2from thecultivated peanut accessions. The full-length of sequences ShgDGAT3-1and ShgDGAT3-2 from Shanhua7are1663bp and1686bp respectively and there are one intrones in each of thesequence. A total of68sites and one sites of endonuclease recognition variations wereidentified between the two sequences.（2） Molecular evolution analysis of DGAT3in peanutThe twelve sequences of gDGAT3-1from the twelve cultivars share99.9%identity innucleotide acid level and the twelve sequences of gDGAT3-2are identical. One DNAsequence of DGAT3was obtained respectively from six wild species. The phylogenetic tree,based on sequences ShgDGAT3-1, ShgDGAT3-2, AdurgDGAT3, AcorgDGAT3, AvilgDGAT3,AcargDGAT3, AipagDGAT3and AbatgDGAT3, show that ShgDGAT3-1grouped togetherwith AvilgDGAT3, AcargDGAT3and ShgDGAT3-2grouped together with AipagDGAT3.（3） Cloning and sequence analysis of SAD in peanutTwo isoforms of SAD cDNA were isolated and named as FhgSAD-1and FhgSAD-2.Comparison of the genomic sequences with cDNAs revealed that there were2introns in theSAD genomic sequences. Sequences alignment showed that the similarity between FhgSAD-1and FhgSAD-2in nucleotide acid level was over97.5%, with69different sites in total,including62SNP sites and6variation sites of endonuclease recognition. The cDNA sequencesimilarity was98.6%. Deduced amino acid sequences revealed that only one differenceoccurred in serine gathering area of¹⁷PSSSSSSSSSSFSL³⁰. gSAD-1shared99.9%nucleotidesequence homology with gSAD-A, while gSAD-2was the same as gSAD-B.（4） Construction of FhrSAD-2expression vectorThe SAD gene was cloned into pEASY-T1vector and then sequenced. The recombinantplasmid pEASY-T1and plant expression vector pGBVE were digested by Hind Ⅲ and Pst I,recover the aimed1500bp fragment and the longer of pGBVE. The result through the PCRdetection and enzyme cut identification proved that the SAD gene was directionally insertedinto the normal vector of pSAD-2and the expression vector of FhrSAD-2gene wasconstructed.（5） Acquirement the positive transformed plants of antisense ahFAD2BThe antisense ahFAD2B was introduced into peanut cultivar and213Kan-resistanceplants were obtained.34T₀individual plants and71T1individual plants from7plant strainswere obtained.13T0individual plants and19T₁individual plants were positive verified byPCR analysis and gain their seeds.