Identification, Characterization and Expression of the Key Enzyme Genes in Biosynthesis of Polyunsaturated Fatty Acids in Pavlova Viridis
|Keywords||Viridis Expressed sequence tags cDNA library Membrane protein Polyunsaturated fatty acids Fatty acid desaturase Fatty acid elongation enzyme Green Fluorescent Protein|
Polyunsaturated fatty acids (polyunsaturated fatty acid, PUFA) are those containing two or more double bonds and carbon chain length of 16-22 carbon atoms, straight-chain fatty acids, including eicosapentaenoic acid (EPA), two docosahexaenoic acid (DHA), of which the EPA / DHA has an important physiological role, such as: nutrition strengthening, regulation of lipid metabolism, prevention and treatment of atherosclerosis and cardiovascular disease, regulate the body's immune function, regulate vision and brain development, regulating the central nervous system function, but also can be developed as anticancer drugs. Currently, EPA and DHA Fish Oil is such an important commercial source of PUFA, however, rely solely on the existing fish resources have been unable to meet the growing market demand. In addition, the study found that fish are not really PUFA producers, but through ingestion of PUFA-rich microalgae accumulate in the body after. Marine microalgae is EPA / DHA primary producers, but also from the PUFA than the production of microalgae extract fish oil process is simple, no fishy smell, low cholesterol. Therefore, the use of marine microalgae for the production of EPA and DHA PUFA become a new means. But mostly marine microalgae autotrophic algae, slow growth, cells have low, the training process tingible bacteria, the other on the scale microalgae culture and improving other aspects of cell research is still at the laboratory stage, so the use of micro- large-scale production of algae extract EPA / DHA to meet the needs of society is not reality. PUFA biosynthesis begins C18-PUFA: LA and A LA, through a series of fatty acid desaturase (fatty acid desaturase, FAD) dehydrogenation and fatty acid carbon chain extending enzyme (fatty aicdelongase, FAE) carbon chain extension role in the gradual completion. FAD catalytic dehydrogenation of the fatty acid chain is converted to CC C = C, FAD in each specific position of acyl chain introducing a double bond; FAE is in C18, C22-PUFA carbon chain extension reaction extends a class enzyme complex that catalyzes the donor (acetyl coenzyme A or malonyl-CoA A) into the two carbon atoms in the carbon chain PUFA increase their carbon chain length. With the PUFA biosynthetic pathway and regulatory mechanisms of research, from the molecular level to study the influencing factors of microalgae PUFA synthesis, through the PUFA biosynthesis key enzyme gene cloning, to achieve efficient expression of the gene variant, in order to get a lot of PUFA , to address EPA / DHA resources, reduce production costs, providing a new way. And FAD and FAE in plant genetic engineering, food engineering and fermentation engineering and other fields of broad application prospects is no doubt, in the field of application will also further promote fatty acid synthesis pathway of knowledge and research. In this thesis, experimental materials viridis (Pavlova viridis) is autotrophic microalgae, EPA, and DHA content-rich, respectively of the total fat content of 16% and 9%, is the study of fatty acid synthesis pathway enzymes are associated dehydrogenase and elongation good experimental material. This work and the results are as follows: 1. Studied the culture temperature, light intensity on P.viridis growth and EPA / DHA content were studied to optimize culture conditions. The results show that, P.viridis wide growth temperature range, at 16 ℃ ~ 25 ℃ can grow, but low temperature (18 ℃) in favor of EPA and DHA algal accumulation in vivo by gas chromatography under the conditions of this EPA and DHA contents were 16.25%, 9.226%, compared with 25 ℃ under the conditions of increased 12.45% and 8.226%. Thus confirmed that: Although higher temperatures favor the growth P.viridis, but this lower content of unsaturated fatty acids. P.viridis of autotrophic organisms, when the light intensity is weak, the algae growth arrest, when the EPA and DHA content is low; With the light intensity increases, the algae grow faster, while EPA and DHA content also increased to 16.62% and 6.79%. By GC / MS was measured under the conditions of 18 ℃ light culture P.viridis fatty acid composition of unsaturated fatty acids found P.viridis range, with a variety of unsaturated fatty acids, and the extension dehydrogenase gene cloned gene good test material . (2) total RNA quality directly affects the merits of the library was built, this paper compares the Trizol reagent method, guanidine isothiocyanate-step, CTAB method and other methods based on a kind of groping for P.viridis total effective method for RNA extraction and acid phenol-CTAB modified CTAB method of combining. This method can effectively remove the RNA extraction process of polysaccharides and polyphenols interference resulting RNA quality is higher. Using the modified CTAB method P.viridis total RNA, the cDNA library constructed, the construction of a cDNA library titer of 6 × 10 ~ 6pfu/mL, recombination rate of 98%, into fragments between 250 bp-2.0 kbp, indicating that the The library building a database to achieve requirements. By expressed sequence tag (expressed sequence tags, ESTs) technology P.viridis cDNA library was amplified and sequenced random sequence, the resulting 200 EST sequences reported in GenBank BLAST homology gene pair won many features Genetic information, for the full-length gene cloning and further development of new functional genes microalgae foundation. After comparison found that in 200 ESTs sequences of genes involved in protein synthesis and involved in energy metabolism (including photosynthesis and respiration) gene content is relatively high, accounting for 41% and 20%. But through the EST technology, no filter to extend dehydrogenase gene or gene sequence, indicating ESTs method does not apply to low abundance genes. 3 Use the SMART RACE technology was first cloned P.viridis C20-extension gene (elkj). Use of green fluorescent protein (green fluorescent protein, GFP) reporter gene expression as elkj, C20-extending the cloned gene (elkj) and GFP gene fusion, achieved elkj the expression in Escherichia coli, protein encoded demonstrated elkj integral membrane proteins, and has a C20-extension activity. elkj gene is 945 bp, encoding 314 amino acids, suggesting that the protein size of 34.5 kDa, predictive analysis by TMHMM hydrophobicity, ELKJ seven transmembrane protein, C terminus is located intracellular lysine has a double structure and the endoplasmic reticulum retention signal. By Southern blot analysis, elkj gene P.viridis genome as a single copy. C20-extending enzyme-catalyzed synthesis EPA DPA, EPA to DHA is synthesized from the key steps. After E. coli fatty acid methyl esters by gas chromatography composed of recombinant bacteria E.coli DE3/pwalEL catalytic EPA C20-generated extension of the DPA enzyme activity was confirmed in P.viridis from the conversion of EPA to DHA requires a two-step synthesis (two step conversion). During the experiment found that when elkj gene expression in E. coli, the recombinant bacteria growth than the control strain E.coliDE3/pwalEL main E.coli DE3/pWaldo-gfpe weak, indicating ELKJ expression on host cells produce toxic effects, leading to expressed difficulties to give the desired protein product. Therefore, ELKJ protein expression and GFP fusion protein, by observing the fluorescence of GFP, the expression reflecting ELKJ. The results show that in the confocal microscope to recombinant bacteria E.coli DE3/pwalEL green fluorescence, indicating ELKJ-GFP fusion protein was expressed correctly, ELKJ protein portion is inserted into the cell membrane, the fusion protein is not formed inclusion bodies; by measuring whole-cell fluorescent signal under different conditions ELKJ compare the expression level of the protein to determine the optimal ELKJ expression conditions. The experimental results show that the use of GFP autofluorescence detection of membrane proteins can be used as indicators of a variety of expression conditions for membrane proteins provides an effective method for eukaryotic membrane protein basis for the study. 4 of the first cloned P.viridis Δ4, Δ5 desaturase full-length gene sequence, including exons and introns and the gene promoter sequence. Molecular biology of marine microalgae late start, the genetic information for reference and operational tools rarely, when the clone P.viridis dehydrogenase gene carried out extensive exploration of the extensive building P.viridis cDNA library screening, do not get dehydrogenase gene sequences, instead SMARTRACE technology and SEFA PCR technique successfully amplified Δ4-desaturase gene (pkjDes4) and Δ5-desaturase gene (pkjDes5). pkjDes4 its full-length open reading frame of 1440 bp, encoding 479 amino acids, submitted to GenBank gain accession no.EF486526; pkjDes5 open reading frame length of 1278 bp, encoding 425 amino acids, submitted to GenBank gain accession no.EF486527. After Southern blot analysis, pkjDes4 and pkjDes5 with C20-elongase genes, in P.viridis genome are single copy gene was confirmed in PUFA biosynthesis enzyme genes in the genome were low copy number, the use of a large number of screening a library Comparison of the target gene obtained difficult. 5 Use cloned from P.viridis C20-elongase and Δ4-desaturase gene elkj gene pkjDes4, completed elkj in Pachia pastoris expression and functional verification, and built elkj and pkjDes4 in P.pastoris the co-expression vector for the use of modern biotechnology to produce DHA basis. PPIC3.5K vector using the integrated construct elkj in P.pastoris expression vector pPEL, high-copy integration into the host screening recombinant strain G3, P.pastoris GS115/pPEL analyzed by gas chromatography fatty acid methyl esters, and are displayed with recombinant G3 catalytic EPA produce the DPA C20-elongase activity, completed the elkj in P.pastoris in functional verification, for the use of in vitro synthesis of DHA P.pastoris way to build the foundation. 6 In the expression vector pNZ8148 lactis as a skeleton in the C-terminal fusion GFP, first construct a Lactococcus lactis expression vector pKj-gfp, and using the carrier to achieve a ELKJ in the overexpression of Lactococcus lactis. As NICE Systems (NIsin controlled expressionsystem) rigor and L.lactis membrane protein expression as a host superiority, NICE system is developed to control the expression of membrane proteins ideal system. By laser scanning confocal microscopy nisin (nisin) after induction L.lactis NZ9000/pKj-el green fluorescence, fluorescent cells are not found, indicating that the plasmid stability in the L.lactis. The use of the expression of GFP fluorescence detection ELKJ process, greatly simplifying the membrane protein expression strain screening and purification process.