Cloning And Functional Analysis Of ACCase And PEPCase Genes In Lipid Biosynthesis Of Rape Seeds (Brassica Napus L.)

Vegetable oil is an essential product in our daily life and constitutes a major source of energy its consumers. The seed is the main storage organ of the oil plants. The improvement in the quality and quantity of seed fatty acid have currently become major research topic. Fatty acid biosynthesis in plants takes place within plastids. Acetyl-coenzyme A carboxylase (ACCase) catalyzes the first committed step of fatty acid synthesis, the carboxylastion of acetyl-CoA to malonyl-CoA which supplies the substrates for fatty acids and many other secondary metabolites synthesis. On the other hand, fatty acids synthesis is closely related to protein synthesis. Phosphoenolpyruvate (PEP) from the glycolysis is a common substrate for fatty acid and protein synthesis. PEP can be converted into pyruvate acid by pyruvate kinase. The pyruvate acid generates acetyl-CoA through the catalysis of pyruvate dehydrogenase. The resulting acetyl-CoA is the key precursor of fatty acid synthesis. Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible β-carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate into protein metabolism. PEPC is a cytoplasmic enzyme with versatile functions in plants. Although PEPC are reported to be involved in fatty acid accumulation and nitrogen assimilation, as well as salt and drought stresses, knowledge on the function of PEPC is still limited, particularly on the bacterial-type PEPC. In this paper, the regulation mechanism on fatty acid metabolism by ACCase and PEPC genes was investigated with forward and reverse genetic methods.Firstly, the E.coli heteromeric ACCase was overexpressed in Brassica seeds to accelerate the formation of malonic acid by acetyl-CoA carboxylation, the key step in fatty acid synthesis. The heteromeric ACCase subunit encoding gene accD is a restriction factor. The accD genes from four close-related species of Brassica were cloned and their sequences were analyzed. Then two plant expressed vectors containing the heterogeneous-type E. coli ACCase accD gene (eaccD) and the genes encoding the four subunits of E.coli ACCase were tandemly constructed. The two vectors were further transformed into the "Chaoyou2" rapeseed to investigate the heterogeneity of eaccD and the other three nuclear genes assembled in fatty acid content. The data indicated that an efficient technique for directed genetic improvement of rapeseed oil content was established. In this research, the gene Atppc4which encodes the bacterial-type PEPC in Arabidopsis was knocked out with the artificial mRNA (amiRNA) technology to investigate the in vivo function. The effect of Atppc4on fatty acid metabolism and resistance to salt stress were elucidated. The study expanded our knowledge on the function of the bacterial-type PEPC in Arabidopsis. The main results are as follows:1. The cDNA fragments of accD gene were amplified from young leaves of Brassica napus, Brassica oleracea, Brassica juncea and Brassica rapa by RT-PCR. The lengths of the amplified accD genes were1470,1470,1464and1464bp, which encode489,489,487and487amino acids, respectively. Sequence analysis showed that the cDNA sequences of the four accD genes were highly homologous. The amino acid sequences deduced from the four accD genes contained a similar zinc finger structure and five identical motifs in C-terminal regions. Among them, motif I (GSMGSVVG) and motif II (PLIIVCASGGARMQE) were present in β-carboxyltransferases of all plant species and E coli. Southern blotting analysis demonstrated that there was only a single copy of accD gene in the genomes of the four close-related species of Brassica.2. To investigate the influence of eaccD gene on Brassica fatty acid content, an expression construct was constructed by fusing the Arabidopsis rubisco small subunit (rbcS) transit peptide and the Brassica seed specific promoter napin to eaccD gene. The expression construct was transformed into B. napus via Agrobacterium-mediated transformation and seven transgenic plants were obtained. The eaccD transcript was confirmed by RT-PCR analysis. The data showed that eaccD was specifically transcripted in seeds. This demonstrated that the expression construct can result in the specific expression of eaccD in immature seeds of Brassica. Seed oil and protein contents of the seeds were detected both in the transgenic lines and the control plants. The total oil content of the control seeds was48.01%and the average protein content was25.10%. Whereas the average fatty acid content in the transgenic lines seeds was52.08%, which was approximately8.45%higher than that of the wild plants. The seed oil content increase was followed by the decrease in protein content. The average protein content was6.69%lower than the wild plants. The weight of1000grains also increased in the transgenic plants. These results suggest that the expression of eaccD in Brassica could significantly increase the seeds oil content and seed weight.3. To increase the content of fatty acid of Brassica by transgenic method, a plant expression vector was constructed by adding an Arabidopsis Rubisco SSU chloroplast signal peptide to the5’end of the target gene ACCase and hooked with the seed special expression promoter Napin in series. Transgenic Brasscia(Brasscia napus L.) plants were obtained via Agrobacterium-mediated hypocotyle.11positively plants were obtained. Four target genes were transformed into To Brassica genome. The seed oil contents of the control and the transgenic plants were48.01%and49.59%, respectively, with the transgenic plants being about3.30%higher than the control plants.4. Plant expressed vector Atppc4-amiRNA was constructed and transformed into Arabidopsis with the in floral dip method via Agrobacterium-mediated transformation. The molecular detection was conducted in Atppc4-amiRNA transgenic lines. The maturedamiRNA was dramatically overexpressed in the transgenic plants. The transgenic plants with constitutively expressed Atppc4-amiRNA exhibited substantially decreased accumulation of Atppc4transcripts, whereas other three plant-type PEPC genes, Atppcl, Atppc2and Atppc3were significantly up-regulated in roots. PEPC activity improved about5.1times in roots of Atppc4-amiRNA transgenic lines. This result indicates that transcription of bacterial-type and plant-type PEPC genes in plants interact with each other in plants. The bacterial-type PEPC genes, Atppc4, may play an important role in modulating the transcription of plant-type PEPC genes. There was no significant difference in fatty acid content between the wild-type plants and transgenic lines. Moreover, fatty acid compositions also had no significant changes in the seeds of wild-type plants and transgenic lines. This research suggested that the transcription of Atppc4might be independent of the lipid content in Arabidopsis.5. Down-regulation of Atppc4improved salt tolerance in Arabidopsis. Studies on7-d-old seedling roots showed that transgenic plants exhibited approximately29.0%less growth in the root system than that of wild-type plants. However when these7-d-old seedlings were moved to a1/2MS medium containing150mM NaCl and incubated under the same conditions for another week, the transgenic plants showed approximately16.3%less root growth than of the wild-type plants. These results showed that salt treatment could partially relieve the inhibition of root growth caused by Atppc4suppression.