Study On Molecular Regulation Of PUFA And TAG Biosynthesis In Nannochloropsis Oceanica

Owing to the prominent contradiction of exhausting fossil fuels and growingenergy demands, the development and utilization of renewable energy has beenextremely urgent. In recent years, there has been a great progress in this field.However, raw materials have still been the "bottleneck", which greatly restricts thedevelopment of biodiesel. The first generation and second generation biodiesel bothcome across the problem that they both have an excessive demand for large amount ofgrains and arable lands.At present, microalgae as the third generation biodiesel is favored by researchersall over the world. Firstly, microalgae can grow in oceans, rivers and lakes withoutoccupying any arable land. Secondly, they can effectively utilize solar energy forautotrophic photosynthesis and lipid enrichment compared with land plants. Thirdly,the growth cycle of microalgae is quite short. Microalgae have been considered as anexcellent choice of biodiesel for high growth and mass cultivation. In addition,microalgae can synthesize polyunsaturated fatty acids （PUFA） and other highvalue-added products, which can effectively share the cost of biodiesel.Genus Nannochloropsis is one of class Eustigmatophyceae. Nannochloropsis isrich in EPA and oil and is expected to be the highest potential species to producebiodiesel. Nonetheless, the biodiesel based on Nannochloropsis is still faced with theproblem of high cost during the mass cultivation. Recent years, the transformation ofNannochloropsis by means of transgenic technologies, aims to acquire an engineeringspecies of excellent qualities and therefore remarkably reduce the cost, which turnsout to be of great importance.Based on the consideration above, the design of this experiment is as followed:according to the previous results of whole genome sequencing of Nannochloropsisoceanica LAMB0001, key genes involved in the biosynthesis pathway of PUFA and TAG are analyzed by bioinformatics method. And then the transformations areconducted by means of genetic engineering from the following perspectives. Firstly,knocking out one key enzyme gene in the pathway of β-oxidation, which iscompetitive with TAG synthesis by homologous recombination; secondly,overexpressing the key enzyme genes both in the biosynthesis of PUFA and TAG. Weattempt to construct an eminent species with an abundant content of TAG and PUFA.First and foremost, we identify4kinds of desaturase （6genes） along thebiosynthesis route of the PUFA; ACS which is responsible for activating fatty acid,amounting to12genes; DGAT, ME and acetyl coenzyme A synthetase along thebiosynthesis route of the TAG, amounting to10genes,2genes and2genesrespectively.We preliminarily analyzed the screened genes:1, We chose the ACS6gene, responsible for the synthesis of the ACS andproviding substrate for the β-oxidation, as the target gene of Homologousrecombination and attempted to primarily explore the Homologous recombinationsystem. First, we acquired the expression cassette and a pair of homologous arms byPCR amplification and then got the “composition segment”（arms1-ble-arms2） bylinking the three segments together. After that, the “composition segment” wastransformed into N. oceanica by electroporation. Positive clones were screened in thef/2solid medium containing Zeocin.100clones were selected and their genomeDNAs were respectively extracted. ORF of ble gene was amplified. The statisticresults reveal that wide type N. oceanica lacked ble gene, while98%transgenic N.oceanica included ble gene. However, with primers from the outsides of ACS6gene,the PCR result indicated the stripes of both the wild type and transgenic ones are same.In other words, although exogenous DNA has been integrated into the genomes of N.oceanica, the homologous recombination did not happen in the target location.Afterwards, we selected No.24transgenic strain and analyzed the randomintegration sites in genome with LA PCR. Through a variety of comparison andanalysis, we concluded that there were at least two random integration sites in No.24, Scaffold8₂（AEUM01005401.1） and Scaffold123₁1（AEUM01000672.1）. Fromthese results, we can firstly verify that exogenous genes are assuredly integrated intothe genomes of N. oceanica. Furthermore, the efficiency of random integration issubstantially high, which can be employed to achieve the overexpression of theexogenous genes in N. oceanica.2, Δ6desaturation enzyme gene （NoD6） and Δ12desaturation enzyme gene（NoD12） in N. oceanica, along with the Δ5desaturation enzyme gene （PtD5） and Δ6desaturation enzyme gene （PtD6） in P. tricornutum were picked as the target genes.We expected to dramatically raise the PUFA content in N. oceanica by theoverexpression mentioned above. Three DGAT genes from P. tricornutum and onefrom A. thaliana are referred to as PtDGAT1、PtDGAT2、PtDGAT3、AtDGATrespectively. DGAT is responsible for catalyzing the last step in the process of TAGsynthesis. NOLACS gene was cloned previously and verified that its products wereengaged in the biosynthesis of TAG. These genes were utilized to significantly raisethe oil content in N. oceanica by overexpression.At first, the cDNA full lengths of the9genes were cloned and then constructedinto the skeleton of the expression vector pCB801-MCS. These recombinant plasmidswere transferred into N. oceanica cells by electroporation.120colonies were selectedand12positive clones were eventually confirmed by PCR amplification, whichderived from5genetic experimental groups--NoD12、NoD6、PtDGAT1、PtDGAT2and AtDGAT. Further researches consisted of growth curve, growth rate and therelationship between total lipid/PUFA and transcriptional level of the transferredgenes which was determined by real time PCR. N. oceanica strains with high lipid orPUFA content were obtained eventually.In general, this study can make reasonable contribution to the revelation of thecomplex PUFA and TAG biosynthesis pathways and provide references to theregulation of gene expression and genetic transformation in N. oceanica. These effortshave important theoretical significance and broad application prospects.