Increasing Astaxanthin Production In Chlorella Zofingiensis By Genetic Modification

Astaxanthin is a secondary carotenoids and has the ability of superior oxidation resistance,quenching singlet oxygen or free radicals,preventing the collective aging,and enhancing immunity.It also has the functions,such as anti cancer,prevention of cardiovascular diseases,and so on.In food industry,it is widely used in food,medicine and health care products.Chlorella zofingiensis contains astaxanthin,and has the advantage of fast growth,high biomass concentration and easy culture etc..But it has the disadvantage of the low astaxanthin content in cells.Methods of application of genetic engineering to improve the astaxanthin productivity become a research hotspot in recent years.In this study,we developed Agrobacterium mediated methods for the genetic modification of Chlorella zofingiensis.First,we constructed a reporter vector of GFP and transformed into Chlorella zofingiensis for establishing the transformed method.After transformed method establishment,we followed construct the expression vector of beta carotene ketolase gene which the gene from Haematococcus Pluvialis for improving the astaxanthin content in Chlorell cell.The contents and results of this thesis are as follows:1.We study the effect of carbenicillin on Agrobacterium and Chlorella zofingiensis the results shown that carbenicillin inhibited the growth of Agrobacterium at the concerntraion of 200mg/L,which was not affected on Chlorella zofingiensis growth at that level.Next,Study on Chlorella zofingiensis sensitivity to G418 in BBM liquid or solid medium were monitored.The screening concentration of G418 is 1mg/L and2mg/L in BBM liquid and solid medium,respectively.According to the study,we choose G418 concentration in liquid medium as 1mg/L and carbenicillin concentration of 500mg/L for screening antibiotic concentration.However,the concentration of G418 and carbenicillin in solid culture were set for 2mg/L and 500mg/L,respectively.2.Transform GFP as a reporter gene.We use the plant expression vector PBI121,whose promoter is CaMV35 S and the selection marker is NPT II.Using Agrobacterium mediated transformation method,we established genetic transformation system of Chlorella zofingiensis.The results shown that GFP gene recombinant in Chlorella zofingiensis at DNA or RNA level confirming by PCR and RT-PCR,respectively.At protein level,we monitored GFP fluorescence under the laser scanning confocal microscope(LSCM).After 200 days of culture,GFP fluorescence was still observedunder LSCM,which means stable positive transformant were obtained.3.Construct PBI121:: 35S:: PDSSP-BKT vector.We used overlap PCR fusion PDS signal peptide gene and BKT beta carotenoid ketolase gene in reading frame for evaluation BKT expression efficiency.PDS signal peptide gene is from tomato and codes the enzyme which converts phytoene into zeta-carotene via the intermediary of phytofluene by the symmetrical introduction of two double bonds at the C-11 and C-11'positions of phytoene.4.With Agrobacterium mediated transformation method,PDSSP-BKT fusion protein gene was tranformed into Chlorella zofingiensis,and positive clones were screened.We extracted astaxanthin from Chlorella zofingiensis and determined its concentration by HPLC.The results shown that under the same culture condition,the astaxanthin content in transformants was higher than that in the wild type which is about 63.5%.