Study On The Regulation Of Lipid Metabolism By The Dof-type Transcription Factor Of Chlamydomonas Reinhardtii

Microalgae biodiesel becomes one of the hottest research work as its advantages in environmental protection, recyclability and the short growth cycle of raw materials. Using genetic engineering to control lipid synthesis and metabolism relevant genes is an important way to improve the regulation of microalgae oil accumulation. Dof-type transcription factor is a specific transcription factor of plants, because it has a mono-zinc finger conserved sequence,called the Dof(DNA binding with one finger). Dof proteins regulate metabolic pathways of plants through regulating the expression of the key gene in carbon and nitrogen metabolism,and in the higher plant expressing the gene of Dof-type transcription factor can enhance the lipid content. In this study, Glycine max Dof-type transcription factor gene(gdof) and Chlamydomonas reinhardtii Dof-type transcription factor gene(crdof) were inserted into C.reinhardtii CC849 by genetic engineering. The target genes can be regulated by an inducible heat shock protein promoter, and over-expression of gdof and crdof in C. reinhardtii was demonstrated to be able to increase the lipid content by real-time PCR and GC-MS. Moreover,the expression levels of eleven fatty acid metabolism associated genes in transgenic algae were analyzed by real-time fluorescence quantitative PCR. The relation between Dof genes and the fatty acid metabolism associated genes was further investigated, suggesting that the increase of fatty acid accumulation was caused by the influence of Dof genes on the fatty acid metabolism associated genes. The results are described as follows:1. The Dof11 gene sequence of Glycine max was obtained from the NCBI, according to the codon bias of C. reinhardtii, to compose the gene c-gdof, while it is the first successful cloning of c-crdof with C.reinhardtii CC849 cDNA as a template. Their length are 960 bp and1875 bp respectively. The c-gdof or c-crdof gene fragment was ligated to the pJD vector to generate the constructs: pJD-c-gdof and pJD-c-crdof. Then these constructs were transformed inti Chlamydomonas reinhardtii by bead milling. The transformants Tranc-crdof-12 and Tranc-gdof-60 expressing high levels of Dof genes were selected by BODIPY staining screeningand qPCR. Expression levels of c-crdof and c-gdof genes in transgenic algae were analyzed by real-time fluorescence quantitative PCR. The results showed that: after heat shock treatment, c-crdof gene expression of transgenic algae Trancr-dof-12 showed the highest increase of 3.51 folds, while c-gdof gene expression of transgenic algae Tranc-gdof-60 showed the highest increase of 4.12 folds.2. To better understand the reason why over-expression of Dof-type transcription factor increases the oil content, the expression levels of 11 genes encoding the enzymes associated with the metabolism of fatty acid in transgenic algae were examined by real-time fluorescence quantitative PCR, i.e., BCC1, BCC2, ACP1, SQD1, SQD2, MGD1, DGD1, FAT1, CIS1, ACS1,and PGP1. We found that the expression levels of the 11 enzymes compared with the control group CC849 varied. The significant changes are as follows: The expression of enzyme in transformants Tranc-crdof-12 showed that, compared with the expression of wild-type, the expression levels of BCC1, FAT1, SQD1, DGD1, MGD and PGP1 increased by 3.42, 5.72,2.1, 7.1, 2.11 and 2.40 folds respectively; while the expression levels of ACS1 and CIS2 decreased by 1.5 and 0.92 folds The expression levels of enzymes in transformants Tranc-gdof-60 showed that, compared with the expression of wild-type,FAT1,DGD1, PGP1 increased by 4.21,1.80 and 3.85 folds, while ACS1 decreased by 1.0 folds.These results indicate that over-expression of c-crdof and c-gdof can influence expression of the key genes in fatty acid metabolism.3. The total fatty acid content and the components in transgenic algae and CC849 were analyzed by GC-MS. We found that transgenic algae contained the highest fatty acids were named Trancr-dof-12 and Tranc-gdof-60, which respectively containing c-crdof and c-gdof. After heat-shock(40?) once, the fatty acids of Trancr-dof-12 and Tranc-gdof-60 were increased by23.24% and 27.79% respectively. Furthermore, after heat-shock(40?) for three times, the fatty acids of Trancr-dof-12 and Tranc-gdof-60 were increased by 61.91% and 71.42%respectively. In Trancr-dof-12 and Tranc-gdof-60, the fatty acid contents of C15: 0, C16: 0, C16: 1,C18: 1t, C18: 3n6 were also remarkably increased. The results suggest that the fatty acid content was significantly increased when over-expressing c-crdof and c-gdof.In summary, the over-expression of c-crdof and c-gdof genes in C. Reinhardtii could increase the oil content, shedding a new light for improving the productivity of microalgae oil.Meanwhile, this work can not only benefit the better understanding of the influence of other metabolic pathways on algae lipid metabolism, but also provide a basis for the further studies on the regulatory roles of transcription factors in algae lipid metabolic pathways.