Optimization Of Genetic Transformation System Of Dunaliella Salina And Modification Of Genes Associated With Oil Synthesis

Dunaliella sallina is a light-autotrophic and single-cell eukaryotic green algae with non-fibrillar cell wall,which synthesizes ?-carotene and lipids at high levels.It is a good resource for the production of natural ?-carotene,high-quality biofuels,and other raw material.In particular,D.salina can grow normally in high-salt environments.Large-scale culture of such algae is not easy infestigated by diseases and insect pests.It has become an important special biological resource for modern agriculture.The establishment of a high-efficiency genetic transformation system of D.salina and the precise modification of target genes can accelerate the directional breeding of the excellent germplasm having breakthrougf traits,and promote the establishment of genetically-modified D.salina strains as platforms for green production of high value-added natural compounds.In this study,D.salina YC-011 strain was used as the host two important enzyme genes,DGAT1a and CrGPAT involved in the biosynthesis of triacylglycerol(TAG)were selected as the targets for modification.the genetic transformation system was optimized,respectively,by electroporation and particle bombardment.Two exogenous target genes were,respectively,introduced into the D.salina cells.The target gene was designed to overexpress in the algal cell,aiming to increaseTAG synthesis.Such transgenic D.salina can be developed as an excellent microalgae for industrial production.The main results obtained in this study were described as the followings:1.Cellular morphology and 18S rDNA molecular identification confirmed the micro alga strain YC-011 previously isolated from Yuncheng Salt Lake,is a new strain of Dunaliella sallina.2.A sterile culture system of D.salina was established by applying combination of 50 ?g/mL chloramphenicol,100 ?g/mL ampicillin and 100 ?g/mL cephalosporin for three times of sterilization and five times of subcultures.3.The expression vector carried the herbicide glufosinate-selective marker(BAR gene).The sensitivity of D.salina cells to glufosinate was quantitatively detected.The glufosinate concentrations used for the positive transformant selection were identified as 20 ?g/mL and 40 ?g/mL,respectively,in solid and liquid cultures.4.An optimized genetic transformation system of D.salina by electroporation was established:D.salina cells cultured for 7 days were the most suitable receptors.The parameters include plasmid concentration of 6 ?g/mL,the shock parameters at 0.4 kv for 4 ms.The transformation rate of D.salina was up to 2.63‰,which is 1.14 times higher than that of the electric shock described previously.5.An optimized genetic transformation system of D.salina by gene gun was established.D.salina cells cultured for 7 days were chosed as receptors.The bombardment parameters include the target distance of 6 cm,and the bombardment pressure of 1300 psi.Each bombardment can generate the transformation rate of 0.13‰.6.VgDGATla cDNA from the high-oil plant Vernonia encodes the diacylglycerol acyltransferase(DGAT1)that catalyzes the final acylation reactionin TAG synthesis.The CrGPAT cDNA from the model microalga Chlamydomonas reinhardtii encodes the glycerol-3-phosphate acyltransferase(CrGPAT),which is the first acyltransferase in TAG biosynthesis.Here,expression vectors of CrGPAT and VgDGATla were successfully constructed,respectively.7.GUS detection and other molecular tests showed that the target genes VgDGAT1a and CrGPAT have been successfully transferred to Dunaliella receptor cells and also highly expressed.8.Physiological and biochemical analysis showed that the lipid content of wild type D.salina was 15.3%,and the VgDGAT1a and CrGPAT transgenic D.salina were 36.9%and 34.8%,respectively.Notably,the overexpression of these two rate-limiting enzyme genes can increase the oil accumulation in microalgal cells.In particular,the increase of oil content did not cause a decrease in protein and carotenoid content and other unfavorable phenotypes.The results of these studies can be applied to the development of microalgae's high-efficiency genetic transformation system,the genetic modification of lipids and other metabolic pathways,and the genetic construction of novel microalgae that can be programmed to control the synthesis of target products.