Cloning And Functional Analysis Of The PDAT1 Genes In Brassica Rapa

Brassica rapa is an important oil crops, which originated in China, and it has been cultured with a long history in our country. Triacylglycerols(TAGs) are the main ingredient in seed oils, which are mainly accumulated in the process of seed development.Furthermore, some amounts of TAG are also accumulated in the vegetative tissue such as petals, anthers, pollen grains, stems and leaves. On the one hand, TAG can provide nutrients for human and animals; on the other hand, it also can be used as chemical materials and a source of biomass energy. Therefore, improving the yield and quality of TAG is one of the main goals of oil crop breeding and energy crop breeding. In addition to Kennedy pathway for TAG synthesis in plants, there also has an acyl-Co A-independent pathway, in which phospholipids are used as acyl donors and diacylglycerol(DAG) is acyl acceptor. This reaction is catalyzed by phospholipid diacylglycerol acyltransferase(PDAT). When DGAT1 gene is mutated, TAG synthesis throught PDAT1 pathway is 70% of the wild type in Arabidopsis seeds. PDAT1 catalyzes the formation of TAG and more unsaturated fatty acyl groups, in particular C18:3 fatty acyl, is transferred to sn-3 position. PDAT, as well as other enzymes, balances of the accumulation of free fatty acid in other tissues. There is one copy of PDAT1 in Arabidopsis genome. How many duplicated PDAT1 genes functionally express in the ancient polyploidy Brassica rapa, whice is still an open quesiton. Therefore, research focused on the function of Brassica rapa PDAT1 is of great significance.Haoyou No.11, a cultivar of Brassica rapa, was used as experimental material in this study. Based on the analysis of Brassica rapa genome, two duplicated PDAT1 genes were identified from Brassica rapa. Bioinformatic analysis was carried out and their functions were verified in Arabidopsis. Main results are as follows:(1)Two PDAT1 coding regions were obtained from Brassica rapa,named Br PDAT1-1 and Br PDAT1-2. The lengths of coding regions are 2007 bp and 1794 bp respectively. Two sequences code peptides with 668 amino acids and 597 amino acids respectively. Sequence alignment by DNAMAN indicated that similarity of Br PDAT1-1 and At PDAT1 was 91.08%, similarity of Br PDAT1-2 and At PDAT1 was 82.86%. Signal peptide prediction showed that Br PDAT1-1 and Br PDAT1-2 have no structure of signal peptides at N-terminal. Transmembrane structure domains analysis showed that Br PDAT1-1 had one transmembrane structure domain, while Br PDAT1-2 had no transmembrane structure domain. Both Br PDAT1-1 and Br PDAT1-2 have phosphatidylcholine-sterol O-acyltransferase superfamily conserved domain.(2) Two plant expression vectors which contain Br PDAT1-1 and Br PDAT1-2 respectively were constructed by Gateway techniques which contain Br PDAT1-1 and Br PDAT1-2 respectively, and then transformed into Arabidopsis wild type col-0. Total lipids were extracted from 7-week-old mature rosette leaves of T3 homozygous transgenic lines and wild type col-0. TAGs were separated by using thin-layer chromatography, and TAG content and fatty acid compositions were measured by gas chromatography. According to the results, overexpressed Br PDAT1-1 in col-0 made TAG level in rosette leaves reach to 4.99mg/g on average, 2.43-fold increase compared with the wild type 2.05mg/g; And overexpressed Br PDAT1-2 in col-0 made TAG level in rosette leaves reach to 5.05mg/g on average, 2.45-fold increase compared with the wild type 2.05mg/g. the contents of unsaturated fatty acid(C18:1, C18:2, C18:3)in rosette leaves of Br PDAT1-1 or Br PDAT1-2 lines were increased. The ratio of unsaturated fatty acids and saturated fatty acids in Br PDAT1-1 transgenic lines was 1.44 on average compared with the 0.76 in wild type. The ratio of unsaturated fatty acids and saturated fatty acids in Br PDAT1-2 transgenic lines was 2.1 on average compared with the 0.76 in wild type.In summary, two Br PDAT1 s show difference in protein structure and both have the function for improving the TAG contents in leaves and improving unsaturated fatty acids in TAGs.