Study On The Gene Transformation System Of Schizochytrium And Application In The Pathway Of DHA Synthesizing

Docosahexaenoic acid (DHA) as an important ω-3polyunsaturated fattyacid(PUFA), has positive effects on development of intelligence and visual acuity andalso has bioactivities on diseases remedies, such as cardiovascular, cancer andthrombosis. Schizochytrium is a marine fungus with rich DHA, which is used as acommercial source for DHA. Study the pathway of DHA synthesis is esssential toimprove the production of DHA. So the paper is concentrated on sequence analysis of18srDNA, optimum of gene transformation system, cloning of pks genes and genemutation in Schizochytrium. The works lay bases for studying the pathway of DHAsynthesis in Schizochytrium.Firstly,18S rDNA genes with length between1751bp and1758bp were clonedfrom Schizochytrium limacinum OUC88and its10derived strains by PCR. Bysequencing and blasting with the18S rDNA sequences from GenBank database, theidentity of S. limacinum OUC88and its derived strains was between98%and99%with Schizochytrium sp FJU-512(GenBank No.AY758384),96%with S. limacinum（GenBank No. AB022107）and93%with S. mangrovei（GenBank No.DQ100293）,respectively. By analysis of the sequence and construction of the phylogenetic tree,the results showed that variation in mutation was less than that in different species.Sequence analysis of18S rDNA is an effective way to identificate the mutants.Secondly, as a basis for optimum of the gene transformation system, thesensitivities of Schizochytrium limacinum to seven antibiotics were reported hereincluding Zeocin, Amphotericin B, G418, chloramphenicol (Cm), ampicillin (Amp),kanamycin (Km) and streptomycin (Str). The results showed that S. limacinum hadthe most sensitivity to Zeocin and then to Amphotericin B, G418and Cm insequences. The survival rate of S. limacinum was only2.10-2.79%(P<0.01) while Zeocin’s concentration was at2.5-4μg/ml. The survival rate of S. limacinum culturedwith Amphotericin B, G418and Cm was <5%(P<0.01),2.08%(P<0.01) and13%(P<0.05) respectively under the higher concentration of each antibiotics which was14-20μg/ml,140μg/ml and25.5-68μg/ml. The results indicated that S. limacinumwas not sensitive to Amp, Km and Str since high content of these antibiotics(50-300μg/ml) can not inhibit the growth of S. limacinum and its survival rate wasstill kept at>80%(P>0.05). This study lays foundation on choice of selective markersfor genetic engineering of Schizochytrium.Thirdly, pks1（3908bp）and pks2（4272bp）genes were cloned from S. limacinumOUC168according to pks gene sequences from GenBank database. The genetransformation system was studied in S. limacinum OUC168by using pks1and pks2as the sites for homologous recombination. Four vectors （pTEF1/Zeo-pks1-U-D、pTEF1/Zeo-pks1-M-D、 pTEF1/Zeo-pks2-U-D、 pTEF1/Zeo-pks2-M-D）with partsof pks1and pks2and the Zeocin resistance gene expression cassette were constructedand transformed into S. limacinum OUC168by electroporation and the transformantswere then selected on Zeocin-containing plates. The integration was confirmed byPCR analysis and Southern blotting. The mutants are as follows:1④（pTEF1/Zeo-pks1-U-D mutant） missed2452bp of pks1;2③and2④（pTEF1/Zeo-pks1-M-D mutants）are inserted1500bp of zeocin gene;3①and3④（pTEF1/Zeo-pks2-U-D mutants） are missed2679bp of pks2;4③（pTEF1/Zeo-pks2-M-D mutant）is missed1132bp of pks2. Because of missing orinserting pks gene, the mutants are vrey different from S. limacinum OUC168. Forexample: the cells are smaller and without fat particles; some mutations have specialsmell; the growing rate becomes slowly and2④is found almost stopping growing;the biomass(6d) is lower than a half of the control-S. limacinum OUC168and thelowest is only0.5g/L; the DHA content and total fatty acids production(6d) arealmost0g/L.The paper lays basis for studying the pathway of DHA synthesis and the functionof some leading enzymes of Schizochytrium. And it also provides a new way forimproving the production of DHA by using genetic engineering.