Cloning Of Genes Related To Biosynthesis Pathways Of Polyunsaturated Fatty Acid And Their Expression In Synechocystis Sp. PCC6803

Polyunsaturated fatty acids (PUFAs) are essential for nutrition and medicine, which have become the hot point for research and industry development in recent years. Traditional polyunsaturated fatty acid products mainly come from fish oil and few shellfish, but overfishing has caused serious environmental and resource problems, and the PUFAs have the problems because of complex separation process and expensive cost. Exploring other ways to develop new commercial productions of PUFAs to substitute the convential resources has become the focus, and utilizing the gene engineering method to produce PUFAs is a new effective means.In this paper, key enzyme genes of fatty acid biosynthetic pathway were cloned and the corresponding expression vectors of peanut, cyanobacteria and Escherichia coli for genetic transformation were constructed and transformed. The main achievements of our studies are as follows:1. Cloning and expression analysis of Acyl-ACP Thioesterases from Arachis hypogaea L.(1) Peanut cultivar’Luhua14’was used in this study. A cDNA fragment of AhFatA which has1650bp was cloned by RACE and Bioinformatic methods. Sequence analysis shows that the open reading frame (ORF) encodes372amino acids, with a calculated molecular mass of40kDa, and the gene has no intron sequence. In addition, a1.2kb cDNA fragment of AhFatB1was amplified using RT-PCR method. Sequence analysis shows that it encodes413amino acids with molecular mass of45.47kDa, a2996bp DNA fragment was isolated and the genomic DNA sequence has six exons and five introns. The subcellular localization analysis indicates that both AhFatA and AhFatB1are localized in cytomembrane, cytoplasm and also in nucleus. The expression patterns of AhFatA and AhFatB1were detected by semi-quantitative RT-PCR and real-time quantitative RT-PCR in different tissues and seeds in different stages, the results showed that the AhFatA transcripts were detected most strongly in seeds of wild-type A. hypogaea L. and were higher at60days after pegging (DAP); the AhFatB1transcripts are detected most strongly in stems and are highest at70DAP, which demonstrates that Fat plays a very important role in fatty acids composition and accumulation of peanut seeds. To further identify the functions of AhFatA and AhFatB1, the sense and antisense plant expression vectors driven by35S promoter and NapinB promoter were constructed and introduced into peanut genome by Agrobacterium-mediated transformation respectively. The transgenic peanuts were obtained and examined by PCR, the verification experiment is in progress. This study will provide new germplasm resources and technical guidance for genetic engineering and research in peanuts.(2) The prokaryotic expression vectors of GST-AhFatA and GST-AhFatB1were constructed and expressed in E. coli BL21(DE3) and the fusion protein was obtained. The transformants cultured at37℃and25℃were collected and dryed, then we measured the fatty acid by gas chromatography (GC). The results showed that AhFatA can increase the contents of palmitoleic acid and oleic acid effectively and AhFatB1can increase the contents of myristic acid, palmitoleic acid and oleic acid effectively. These results indicates that Acyl-ACP Thioesterases of peanut were at least partially responsible for improving the synthesis of upstream substrate of PUFAs.(3) The homologous recombinant vectors of Acyl-ACP thioesterases of peanut driven by psbA2promoter were constructed and transformed into Synechocystis sp. PCC6803. The gas chromatography analysis shows that the content of palmitoleic acid and stearic acid increased obviously in transgenic cyanobacteria.2. Expression studies of fatty acids desaturases of ω-3biosynthetic pathway in Synechocystis sp. PCC6803(1) In this paper, the△15and△6fatty acid desaturases of Synechocystis sp. PCC6803were cloned and the Gibberella fujikuroi bifunctional△12/△15fatty acid desaturase and the Mortierella alpina A6fatty acid desaturase were optimized and synthesized. The psbA2promoter of Synechocystis sp. PCC6803was cloned and reconstructed with fatty acid desaturases,10recombinant plasmids were constructed and transformed Synechocystis sp. PCC6803.(2) The gas chromatography analysis shows that the content of a-linolenic acid and stearidonic acid in transgenic cyanobacteria increases significantly and reduces γ-linolenic acid content and ω6/ω3fatty acid ratio.In this study, some genes about fatty acid synthesis were cloned and transformed into Synechocystis sp. PCC6803, then the transgenic cyanobacteria that high polyunsaturated fatty acids were obtained. This study lays foundation for improving the fatty acids content of cyanobacteria and providing the theoretical basis and experimental programs for large-scale production of EPA and DHA.