Transformation Of Gpd1 Gene Into Chlamydomonas Reinhardtii By Agrobacterium Tumefaciens-Mediated Transformation

Nowadays,because of the exhausting traditional fossil fuels and deteriorating environmental problems,bioenergy,especially algae biodiesel,has been concerned by worldwide scientists.In addition,algae biodiesel has lots of advantages,such as high oil content,high biomass,easy to cultivate and harvest,do not occupy the cultivated land,etc.modem biotechnologies can be used to strengthen the microalgae lipid metabolism pathway,so that the increased oil content has the ability to make algae biodiesel more competitive.Chlamydomonas reinhardtii,a eukaryotic single cell algae with clear genetic background,is used in the field of biofuel and lipid metabolism genes,research.Glycerol-3-phosphate dehydrogenase 1(gpdl)regulates the activity of the key enzyme glycerol-3-phosphate dehydrogenase(GPDH)in plant lipid metabolism.GPDH not only promotes the synthesis of plant lipids but also determines the synthesis of Triglycerides(TAG)in Chlamydomonas reinhardtii.With the exogenous gpdl studied in Chlamydomonas reinhardtii,the TAG metabolism and the regulation of lipid accumulation conditions in Chlamydomonas reinhardtii are easy to be expounded.Studies have shown that the overexpression of exogenous gpdl gene in the cell wall defect type algae can increase the oil content of algal cells.However,the selected cell wall defect type algae material causes the limitation of the overexpression study of gpd1 gene.Based on this,the agrobacterium-mediated method was used to overexpress the gpdl gene in Chlamydomonas reinhardtii(with cell wall)to increase algal cell oil content in this study.The recombinant plasmid pRI-Ble-GPD1,constructed with the gpd1 gene cloned from Saccharomyces cerevisiae,was transferred to the FACHB-479 genome by agrobacterium-mediated method with using Chlamydomonas reinhardtii(FACHB-479)as a receptor.Transgenic algae,in which the oil content was increased,was identified through the screening culture,PCR and RT-PCR molecular method.Specific research results are as follows:1.A culture system of Chlamydomonas reinhardtii FACHB-479 is optimized in this study.The culture system is that,TAP medium,2000Lx,25±2?,and several times'shocked at 100rpm for 5min.2.In this study,a binary expression vector pRI-Ble-GPD1 prefer Agrobacterium tumefaciens-mediated transformation of Chlamydomonas reinhardtii FACHB-479 with exogenous yeast gpdl gene was constructed by Cloning the gpdl gene from yeast,construction of recombinant cloning vector pMD-GPD1,construction of recombinant vector pDBle-GPD1 and Construction of recombinant expression vector pRI-Ble-GPD1.Then,the expression vector pRI-Ble-GPD1 was introduced into LBA4404 by freeze-thaw method.After that,the genetically modified LBA4404 was identified to be positive clones,which can transform exogenous gpdl gene to Chlamydomonas,by using amicillin resistance screening system.3.A genetically modified strain FACHB-GPD of Chlamydomonas reinhardtii was constructed by the following procedure.First,the expression vector pRI-Ble-GPD1 was introduced into FACHB-479 by Agrobacterium-mediated transformation method.Second,the positive clones were screened by zeocin resistance,PCR and RT-PCR.4.In this study,a method of oil-Nile red staining is optimized with a final concentration of 40 mg/L of Nile Red/methanol,23?,15min staining in the dark.5.The oil content in Chlamydomonas reinhardtii strain FACHB-GPD cells is 1.3?1.52 times to the wild type FACHB-479 by the fluorescence analysis with Nile red staining and multifunctional microplate reader.