Cloning And Characterization Of Diacylglycerol Acyltransferase (DGAT) Gene In Peanut (Arachis Hypogaea L.)

Triacylglycerols (TAG) are the main storage lipids in most oil plants. Diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) is the rate-limiting enzyme that catalyzes the final step in triacylglycerol (TAG) biosynthesis by converting diacylgycerol (DAG) and fatty acyl-coenzyme A (CoA) into triacylglycero1. Three different types of DGAT protein are identified from plant up to now. DGAT1 and DGAT2 were isolated from many species such as Arabidopsis thaliana, Brassica napus and otherwise, but the third type of DGAT was just detected in peanut. The DGAT proteins of three different types share lower similarity, but all of them have an acyltransferase domain, while the DGAT proteins of the same type appear to be conserved in the C-terminal. The DGAT proteins in plant play an important role in seed development, seed germination, seedling development and leaf senescence.The sequence of Arachis hypogaea DGAT1 (AhDGAT1) gene and Arachis hypogaea DGAT2 (AhDGAT2) gene were undiscovered, then the sequence of GmDGAT1 gene in Soybean and MsDGAT2 gene in clover blossom were used as grounds to abtain the cDNA sequence of AhDGAT1 and AhDGAT2 by homology cloning and rapid-amplification of cDNA ends (RACE) method. In conclusion, partial AhDGAT1 gene sequence of 1480bp and AhDGAT2 gene sequence of 1222bp were obtained. Compared to other DGAT members in plant, the protein encoded by partial sequence of AhDGAT1 gene belongs to DGAT1 family, and the protein encoded by AhDGAT2 gene belongs to DGAT2 family.Two types of AhDGAT2 gene were cloned from Luhua 14, and named AhDGAT2a and AhDGAT2b respectively. Sequence analysis shows that both of them contain a single open reading frame (ORF) of 1005bp and a 3'UTR (Untranslated Region) of 192bp. There are twelve bases differences between the coding regions of the two AhDGAT2 genes. AhDGAT2a encodes a protein of 334 amino acids with an estimated molecular mass of 37.502 kD and an isoelectric of 9.47, while AhDGAT2b encodes a protein of 334 amino acids with an estimated molecular mass of 37.540 kD and an isoelectric of 9.53. Three amino acids differences were detected in the N-terminal between the two AhDGAT2 proteins. The analysis result of TMHMM 2.0 indicates that AhDGAT2 protein contain two membrane spanning regions in the N-terminal. It was found that the polymorphism of the amino acids sequences of AhDGAT2 proteins appears among different peanut species.The expression patterns of AhDGAT1 and AhDGAT2 in different organs of peanut were analyzed by semiquantitative RT-PCR. It was found that the expression level of AhDGAT1 was highest in flower, and that of AhDGAT2 was highest in both flower and leaf. The results displayed that AhDGAT2 may play a more important role in TAG biosynthesis in leaves compared to AhDGAT1. During seed development, both AhDGAT1 and AhDGAT2 expressed higher in early stages. AhDGAT1 gene mainly expressed in 2-4 stages, and came to a head in the third stages, while the expression level of AhDGAT2 was higher in the second phase. This data indicates that the two AhDGAT genes are likely to have different functions in different development phases.To investigate the functions of the two types of AhDGAT2, four types of vectors including constitutive overexpression vector, constitutive antisense expression vector, seed-specific overexpression vector, seed-specific antisense expression vector were constructed. The constructs of pROK II-35S-AhDGAT2a and pROK II-35S- AhDGAT2b were introduced into tobacco and peanut respectively by Agrobacterium- mediated transformation. 18 lines of transgenic tobacco and 7 lines of transgenic peanut were obtained for AhDGAT2a, while 8 lines of transgenic tobacco and 1 lines of transgenic peanut were obtained for AhDGAT2b. The content of fatty acid in leaves of serveral transgenic tobacco lines was detected by gas chromatograph. The results indicated that total amount of fatty acids was increased by 5.37%-22.82%in transgenic tobacco plants compared to the wild tobacco. Furthermore, the content of the unsaturated fatty acid (C16:1,C18:1n9,C18:3n3,C20:1,C22:5n3) were increased significantly, whereas the content of saturated fatty acid (C16:0,C18:0, C20:0,C24:0) stay the same. Then it is supposed that AhDGAT2 may increase the fatty acid content in transgenic tobacco plants.