| Very long chain polyunsaturated fatty acids (VLCPUFAs, defined as C20+fatty acidscontaining three or more double bonds), such as arachidonic acid (AA,20:4Δ5,8,11,14),eicosapentaenoic acid (EPA,20:5Δ5,8,11,14,17) and docosapentaenoic acid (DHA,22:6Δ4,7,10,13,16,19) are essential for human health and nutrition. Human beings can notsynthesize these fatty acids effectively, so dietary supplementation with these fatty acids isnecessary. At the moment, the primary source of EPA and DHA in the human diet is marinealgae oil and marine fish. However, the artificial enrichment of algae in the culture is prone toproduce by-products and toxins. Meanwhile, marine fish stocks are in severe decline as aresult of decades of overfishing. Moreover, pollution of marine ecosystems has resulted in theaccumulation of toxic chemicals in fish, to the point of limiting the benefits of fishconsumption to human health. In consideration of these points, there is an urgent need forseeking an alternative and sustainable source of VLCPUFAs. The possibility of usingtransgenic plants that have been engineered to synthesize and accumulate VLCPUFAs in theirstorage seed oils has been thoroughly investigated over the last15years. The main problem ofthis source is that the yield of EPA especially DHA is relatively low, which is often ccoupledwith the levels of other undesired fatty acids being too high in the transgenic plants. Therefore,to identify enzymes conferring high catalytic activity and strong substrate specificity is one ofthe effective ways to improve the quality and yield of fish oil in transgenic crop plants.In this study, we firstly functionally characterized an Isochrysis galbana Δ5desaturasegene IgD5by expressing it in Arabidopsis thaliana. Then we reconstituted the Phytophthorainfestans EPA biosynthesis pathway in tobacco. The result confirms that the biosynthesis ofEPA in Phytophthora infestans is via the Δ6desaturation pathway. Secondly, we modified thekey enzymes involved in the biosynthesis of EPA/DHA by codon optimization and improved the level of their expression in plants. Thirdly, we constructed a series of auxiliary vectors thatenabled us to successfully build a number of EPA biosynthetic expression vectors consistingof3-4transgene expression cassettes within the same T-DNA. We then tansformedArabidopsis and cotton plants with these EPA biosynthetic expression vectors. EPA can bedetected in both transgenic A. thaliana and G. hirsutum. The main results were as follows:(1) Functional characterization of I.galbana Δ5desaturase gene IgD5in A.thalianaWe isolated a Δ5desaturase gene from the unicellular microalga, I. galbana, namely IgD5.We transformed IgD5into a double transgenic A. thaliana (TMA2) that can alreadysynthesize di-homo-γ-linolenic acid (DGLA,20:3Δ8,11,14) and eicosatetraenoic acid (ETA,20:4Δ8,11,14,17). Triple transgenic plants were identified by PCR and RT-PCR. The total fattyacids composition of the transgenic lines was determined by gas chromatography. Novel fattyacids, AA and EPA were found to be present and accounted for7.04%and2.07%of total fattyacids, representing a conversion rate of68%and60%, respectively, values much higher thanthose for yeast expression. In addition, IgD5did not show a preference for either ω3or ω6substrate. Furthermore, IgD5has strong substrate specificity as no other fatty acid productswere detected other than AA and EPA in transgenic Arabidopsis. These new findings furtherconfirms that IgD5is a gene of preference for the production of fish oil in crop plants due toits high enzyme activity coupled with strict substrate specificity.(2) Functional characterization of P. infestans Δ6and Δ5desaturase gene in N. tabacumPhytophthora infestans is rich in EPA. We cloned two desaturase genes, PinD6andPinD5from this fungus. Bioinformatics analysis showed that both desaturases contained fourconserved domains. By our multigene stacking method, we constructed a plant expressionvector containing PinD6, PinE6and PinD5and transformed it into tobacco. Transgenic plantswere identified by PCR. The composition of the total fatty acids of the transgenic leaves wasdetermined by gas chromatography. Novel fatty acids EPA was identified and it wasaccounted for0.8%of the total fatty acids. This confirms that the biosynthesis of EPA in P.infestans is most probably via the Δ6desaturation pathway. It is well known that AA and EPAare released from germinating spores of P. infestans onto their host plants during infection andthese two fatty acids can regulate the activity of enzymes involved in plant immune response.Therefore, our study towards the reconstitution of EPA with the three key enzymes isolated from P. infestans is very important information for further understanding of the pathogenesishence prevention of P. infestans.(3) The codon optimization of Δ5desaturase gene PinD5from P.infestansThe last step for EPA biosynthesis of both the classic Δ6desaturation pathway and thealternative Δ8desaturation pathway is catalysed by the Δ5desaturase. Therefore, this enzymehas a significant impact on the production of AA and EPA whichever pathway is concerned.Previously, we have cloned a Δ5desaturase gene PinD5from P.infestans, and its enzymeactivity was very low when expressed in yeast. Comparasion on codon usage of the PinD5between P. infestans and S. cerevisiae showed that its5′terminal codons are used at low levelsin S. cerevisiae. We, therefore, optimized the first16codons of PinD5to the codons that areused most frequently in S. cerevisiae. The optimized PinD5was transformed into yeast and itsenzyme activity was analysed. The wild type Δ5desaturase with no codon change were usedas a control.We found that the enzyme activities of the optimized PinD5was doubled that ofthe wild type. Our data provide evidence that codon optimization could impove the expressionlevel of VLCPUFA related genes in yeast.(4) Creation and validation of a widely applicable multiple gene transfer vector systemFunctional identification of genes involved in the VLCPUFAs biosynthesis pathway andtheir reconstitution in oilseed crops is a huge task for genetic metabolic engineering. This isbecause it will need to introduce several desaturase and elongase genes into the plant bygenetic transformation. In order to solve the problem of multi-gene transformation, weconstructed a series of multi-gene transfer auxiliary vectors incorporated in differentpromoters. These include the pASA1(35S), pASA2(Napin), pASA3(Ubi) and pASA4(Actin)vectors which all contain a specific structure AvrII--SpeI-AvrII for gene stacking. We alsomodified the pAUX2vector by exchanging its35S promoter to the seed-specific Napinpromoter to create the pAUX3(Napin) vector in which the XbaI--AvrII-XbaI structure couldbe used for transgene stacking.(5) Application of the multi-gene auxiliary vector system for the construction andproduction of EPA in the plantIn a proof-of-concept experiment, we constructed two plant transformation vectors forEPA production. The first one contains35S promoter, and the second one contains Napin promoter. We transformed A. thaliana via Agrobaterium mediated transformation methodusing these two vectors respectively and obtained transgenic A. thaliana. The total fatty acidsin the seeds of the transgenics were determined by gas chromatography. This confirmed thepresence of EPA in both the35S and Napin constructs transformed plants. The results showedthat all the transgenes had been transfered and expressed in A. thaliana.To further verify the usefulness and stability of the multigene transfer vectors in cropmetabolic engineering we isolated four genes encoding a9elongase, a8desaturase, a5desaturase, and a15desaturase. They were individually cloned into the plant expressioncassette in the pAUX2vector, which consists of the CaMV35S promoter and the NOSterminator. These four cassettes were then linked together into the plant vector pCambia2300that has the kanamycin resistant gene for transformation selection in plants. The recombinantplasmid pCambia2300-4EC was transferred into cotton by Agrobacterium mediatedtransformation method. Transgenic cotton seedlings were first identified by screening thembased on kanamycin-containing media and followed by PCR with gene-specific primers of thefour transgenes. Finally, these transgenics were subjected to gas liquid chromatographyanalysis for their fatty acid composition and the results showed that the contents of AA andEPA were1.0%and5.0%respectively in the leaves of two transgenic plants, indicating thatthe four transgenes were expressed in cotton. Therefore, our data clearly demonstrated that theauxiliary vectors can be used for constructing binary vectors for the stable transformationgene expression in plants. This study will lay a foundation for the production of VLCPUFAs,including EPA and DHA in cotton seed through transgenic technology in the future. |
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