Global Bioinformatic Analysis Of Diacylglycerol Acyltransferases (DGAT) And Functional Identification Of Six DGAT Genes From Phytophthora Infestans

Using genetic engineering to heterologous synthesis very long-chain poly unsaturated fatty acids, VLCPUFAs, in plants is an ideal way to solvethe decline problem offish oil yield and quality. Effort has been focused on the cloning and expression of the microorganism-derived genes for the biosynthesis of EPA (eciosapentanoic acid) and DHA (docosahexanoic acid)in higher plants. This met various degrees of success. However, to produce high enough yield of these VLCPUFAs in trangenetic higher plants remains to be a chanlenge. This is the main obstacle for the commercialization of these VLCPUFA-producing transgenic crop plants. Fatty acids, after synthesed, are stored primarily in seed oil as triacylglycerol (TAG). The yield of VLCPUFAscan be improved if they could be tranfered and used for TAG biosynthesis. Thus, the enzymes that carry out the transfer of fatty acids for the biosynthesisof TAG will be of particular importance for the accumulationof these fatty acids in the seed oil of plants.DGAT (acyl-CoA:diacylglycerol acyltransferase)commits in the final step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme. There are two main types of DGATs, DGAT1and DGAT2, which are the main enzymes responsible for TAG synthesis. In addition, the bi-functional WS/DGAT and cytosol DGAT were also found in some oranisms.DGAT1and DGAT2, which do not share high DNA or protein sequence similarities, are different in their biochemical properties, the subcellular localization and physiological functions. I It has been suggested that they play non-redundant roles in different tissues and in some species for TAG biosynthesis.DGATl andDGAT2have been showed to have preferences on the choice of substrate fatty acids.Using bioinformatics, we conducted a broad survey of fully sequenced genomes of higher plants, fungi and animals, and identified166DGAT genes. We analysised in details of these166genes in terms of the differences of their gene structure, the transmembrane domain(s) of their encoded proteins and their expression patterns in Arabidopsis,soybean, corn, rice, tomato and mouse. The different function between DGAT1and DGAT2was explored from the perspective of evolutionary.To take this further we identified and isolated six DGAT genes from the potato late blight causal pathogen Phytophthorainfestans. Because P. infestans is rich in the long chain polyunsaturated fatty acids EPA (15%of total fatty acid) we reason that the PiDGATs may be prefer the use VLCPUFAs for TAG biosynthesis, therefore they could be used to increase such fatty acids in seed oil in transgenic crops. We first verified their DGAT enzymatic activity by complementation studies in the yeast dgat mutant. We then expressed them individually in Arabidopsis and measured the TAG content in the transgenics. These identified PiDGAT genes could be used for the modification the fatty acids in TAG as well as for elevation of TAG contentin transgenic crops..The main findings are detailed as follows:(1)By a broad survey of fully sequenced genomes,146DGATs were identified from35higher plants where53DGAT1,83DGAT2,5DGAT3(Cytosolic DGAT) and5DGAT4(WS/DGAT) were found. Two DGAT1and12DGAT2were identified in6types of fungi while six DGAT1and DGAT2were identified from each of the6types of animals. This clearly demonstrated that DGAT1and DGAT2are widespread in eukaryotes while DGAT3and DGAT4are specific for the cruciferae of higher plants.(2) The analysis of gene structure, transmembrane domain of the encoded proteins and expression patterns in the5higher plants Arabidopsis, soybean, corn, rice, tomato and the animal mouse showed that DGAT1and DGAT2belong to different gene families which are highly conserved yet very different. Gene chip analysis showed that DGATs are differantially expressed during growth and development between the five different higher plants studied.(3) We cloned6DGAT from Phytophthorainfestans. Bioinformatics analysis showed that these six DGAT belong to DGAT2. So we named the six DGAT as PiDGAT2-1/2/3/4/5/6.(4) The six PiDGATs were individually expressed in the yeast DGAT knockout H1246, which can not grow in the presence of oleic acid. The transgenic yeast harbouring the PiDGATs can return to normal growth in the medium containing oleic acid, indicating that they cancatalyse the generation of TAG in the yeast mutant. Therefore, all six PiDGATs have DGAT enzyme activity. We also measured the TAG contents of the transgenic yeast and showed that transgentic yeast harbouring the PiDGAT2-4produced the highest amount of TAG.(5) We used both constitutive35S promoter and the seed specific Napin promoter to drive the expression of these six PiDGATs in transgenic Arabidopsis. The results showed thatthe TAG content in all the five35S:PiDGATs transgenic lineswas significantly higher than the wild type except35S:PiDGAT2-5line, In transgenic homozygotes of Napin:PiDGATs plants, the seed weights were increased which was consistent with the increase of the TAG content.