Studies On Astaxanthin Synthesis Of Transformed Dunaliella Salina With ?-carotene Ketolase And ?-carotene Hydroxylase

Astaxanthin is a high economic value derivative of beta carotene,which plays a notable role in oxidation resistance in organisms,and therefore it is an important raw material for pharmacy and healthy industries.Dunaliella salina is a saline micro alga,which adepts the environments widely,grows faster and the cost for culture comparatively lower.The alga often accumulates beta carotene,which is a precursor of astaxanthin,more than 10% of its dry weight.However,D.salina is lack of the enzymes for astaxanthin synthesis so that the accumulated beta-carotene is hardly converted into astaxanthin in the alga.The experiment was designed that the exogenous key enzyme genes for astaxanthin synthesis,?-carotene ketolase(bkt)and ?-carotene hydroxylase(crt Z),were transformed into D.salina by bombardment deliver system.The transformants selected by screening might demonstrate that the exogenous genes could be expressed and functioned in D.salina.Meanwhile,a large scale culture of the transformed D.salina as new producer will be great valuable for yield of astaxanthin.The bkt and crtZ genes were cloned from Haematococcus pluvislis by isolated total RNAs and then reverse transcription PCR.The full length of bkt was 1363 bp with a 990 bp ORF encoding 329 amino acids.The full length of crtZ was 1138 bp with an 882 bp ORF encoding 293 amino acids.The pEGAD vector was adapted as an expression vector by fusing cloned bkt and crtZ genes.The recombined vectors were named as pEGAD-bktO and pEGAD-crtZO respectively.The sterilized D.salina culture was obtained by treatment of chloramphenicol and it was found that the optimum concentration was 15?g/mL by gradient concentration screening.The recombined pEGAD-bktO and pEGAD–crtZO were transformed into sterilized D.salina cells by bombardment deliver system.The transformants were screened under the pressure of 3.5?g/ml of glufosinate herbicide.The genomic DNA was extracted from the screened transformants for PCR reactions to detect the transformed genes.The confirmed transformants were treated under high irradiation intensity to induce the synthesis of astaxanthin.The amount of astaxanthin was 3.71?g/g dry weight in transformed D.salina measured by HPLC.