The Study Of The Biochemical Fingerprints Of 6 Dunalialle Species And The Cloning Of ACCase Beta Subunit From Dunaliella Salina

In this study, Dunaliella species saved by Kaiya Biotechnological Corporation of Sichuan University were used. First, bottle culture was taken ,then scale-up cultivation for collecting enough biomass for further experiment.The growth period of 6 Dunaliella cells were measured, and the the growth curve of them were described. No significant difference was detected among the growth rates of the 6 Dunaliella species. Dunaliella entered the logarithmic phase about 140 hours after artificial inoculation, and the stationary phase about 210 hours, which was consistent with other reports. The results showed that each species among 6 Dunaliella species reported here contained significant quantities of one or more nutrients measured in this study. Compared to the frequently reported nutritional components in D.salina, those in D.parva drew more attention in this study. D.parva contained 44.30% lipid of a dry weight, which was much higher than those reported elsewhere. D. bioculata and D. primolecta contained 40.78% and 39.59% of lipid, respectively.The chlorophyll content ranged from 29.23μg/ml to 19.78μg/ml. Ethanol precipitation was used to extract the polysaccharide, and the 3,5-dinitrosalicylic acid colorimetric method was used to determine the content of polysaccharide. The Bradford assay was used to determine the content of protein in the algae cells, but diffrerent ways of sample treatment were done. Wet algae cells obtained from centrifugation were used to test the content of protein in this study, while dry cells through centrifugation and vacuum freeze-dehydrating in other studies.Acetyl-CoA carboxylase (ACCase, EC 6. 4. 1. 2) was identified as biotincontaining enzyme type I . It is the rate-limiting enzyme of synthesis, catalyzes the first reaction of synthesis of fatty acids. Acetyl-CoA carboxylase catalyzes(ACCase) is the carboxylation of acetyl-CoA to produce malonyl-CoA. ACCase is an important enzyme of regulating fatty acid synthesis metabolism.There are two ACCase informs that are encoded by distinct genes. the ACCase cloned from Dunaliella salina in this study. According to conserved motif of the homologous amino acid sequence of ten kinds of algae, a pair of degenerate primers were designed and a cDNA fragment of D. salina with a length of 400 bp is obtained by RT- PCR technique , which has highly similarity with ACCβfrom other species. A 1288 bp full-length ACCβcDNA of D. salina (GeneBank accession No. EF363909) is acquired by 5'RACE and 3'RACE technology . The resulting PCR products were inserted into T- vector then transformed into Top10. Three colonies were selected to determine their sequences. Homologous analysis of the deduced amino acid sequences were performed by BLAST and subsequently compared with GenBank data. Result : Three nucleotide sequences were obtained , of which contain 1288bp coding 349 amino acids. The sequences share high homology with assimilatory nitrate reductase , with identity 84% to NP₀45833.1 (chloroplast Chlorella vulgaris),82% to YP₆35822.1 (chloroplast Oltmannsiellopsis viridis),80% to YP₆36254.1 (chloroplast Pseudendoclonium akinetum) , respectively. Conclusion : The cloned sequence is nitrate reductase cDNA fragment from Dunaliella salina.