Research On Fatty Acid Desaturases In EPA Biosynthesis Of Phaeodactylum Tricornutum And Nannochloropsis Oculata

It is well known that very long-chain polyunsaturated fatty acids (VLCPUFAs) have a number of important nutritional and pharmaceutical values. Dietary supplementation of VLCPUFAs has been found significantly alleviate the symptoms of many diseases. Currently, the principal source of VLCPUFAs is deep-sea fish. However, it is considered insufficient and endangered due to an expanding market and environmental pollution. To exploit sustainable and continuable sources is urgent need. Marine microalgae have been recently shown to be good alternative sources for their ability to synthesis and accumulate great amounts of VLCPUFAs. More and more genes encoding enzymes involved in VLCPUFAs biosynthesis of microalgae, eg. desaturases and elongases, have been identified. Co-expression of such genes in oil crops to produce VLCPUFAs becomes one of the hotspots in the field of plant metabolic engineering. Several foreign groups studied VLCPUFAs biosynthesis in transgenic plants and breakthrough has been made. There is no domestic related reports and it is necessary to do some research work in time. It is useful to demonstrate the mechanism of manipulation of VLCPUFAs production in microalgae and offer molecular basis for the following transgenic research.In this study, two marine microalgae, Phaeodactylum tricornutum and Nannochloropsis oculata were chosen for study of their desaturases. Both of them are rich in EPA and widely used in mariculture. There are some differences in their fatty acid synthesis. EPA biosynthetic pathway of P. tricornutum is more complex than N. oculata. There is a little DHA in P. tricornutum, but no DHA in N. oculata. Several desaturase genes have been cloned from P. tricornutum. However, little has been known about fatty acid biosynthesis of N. oculata at the molecular level until now. Therefore, different methods were used to clone desaturase genes from these two microalgae.According to the previous reports, theΔ5 desaturation catalyzed by theΔ5 desaturase is the key process in EPA biosynthesis. The conversion of 20:4n3 to EPA is almost 100% in P. tricornutum. Though aΔ5 desaturase from P. tricornutum (PtD5) has been identified, it seems that its capacity of converting 20:4n3 to EPA is not as high as anticipatory. So maybe there is a secondΔ5 desaturase in P. tricornutum. To obtain this gene, degenerate PCR, inverse PCR and RACE were performed. As a result, aΔ5 desaturase-like sequence was obtained (PtD5-2). The total sequence is 1561bp with an intron in 303-432bp. The ORF is 1431nt encoding 476 amino acids. The deduced protein sequence contains the typical characters of'front-end'desaturases such as the N-terminal Cyt b5 domain and three conserved histidine clusters with an H to Q substitution in the third cluster. In addition, hydrophobic plot had indicated the presence of distinct four potential transmembrane helices. The protein sequence shares the higher identities with PtD5 and theΔ5 desaturase of Thalassiosira pseudonana (DesO) (51% and 47%, respectively). The identification of this gene is in processing.Though there are some morphological, biochemical and biophysical studies of N. oculata, little has been known at the molecular level until now. Considering this alga has great ecological and commercial value, more efforts should be made to obtain information of its genome, which might help us better understand its various biological processes including fatty acid biosythesis. To generate expressed sequence tags (ESTs) is an efficient way to obtain information on expressed genes. So a cDNA library was constructed using N. oculata cells grown at late exponential growth phase. More than 5, 000 clones were sequenced and 5315 EST reads was obtained. A total of 1960 non-redundant sequences (NRSs) were generated. Only 32.5% of NRSs showed significant similarity (E<1e-04) to proteins registered in GenBank non-redundant protein database. The KOG profile database returned significant hits for 490 NRSs . Analysis revealed that a large proportion of NRSs could be unique to this microalga. The desaturase-like genes were failed to be detected. In addition, it was very strange that no product could be obtained when using degenerate primers to do PCR. It seems that such genes are also be unique to N. oculata.Moreover, the gene expression pattern of PtD5 and PtD5-2 at different growth phases of P. tricornutum was analyzed by Real-Time PCR. The results showed that both of them were most highly expressed at exponential growth phase and then remarkably reduced. However, EPA content of P. tricornutum was highest at the end of exponential phase. Maybe it suggests time is needed during the course from gene expression to product synthesis. Further study will find the real reason. This results offer useful information for research on other microalgae. If the expression of desaturase genes of N. oculata is similar with that of P. tricornutum, maybe the cells grown at exponential growth phase are better for cDNA library construction.In a word, aΔ5 fatty acid desaturase-like gene of P. tricornutum was isolated in this study. If this gene can be proved to be aΔ5 desaturase, P. tricornutum will be the first organism that has two functionalΔ5 desaturase genes involved in EPA biosynthesis. Moreover, the first characterization of EST dataset from Nannochloropsis oculata were also presented in this study. This work set the molecular basis for further studies on N. oculata to better understand its various cellular processes including EPA biosynthesis.