Molecular Identification And Halophilic Characteristics Investigation Of Dunaliella Sp. D3

The genus Dunaliella (Chlorophyta) is a kind of important economicmicroalgae, which can be used to produce many products such as β-carotene and glycerol. Mostspecies of Dunaliella can adapt to an exceptionally wide range of salt concentrations, for example,from0.05to5.50M NaCl. Therefore, many species of Dunaliella are good experimental materialsto study salt-tolerant mechanisms of algae.In this paper, a strain of Dunaliella sp. D3was determined its taxonomy based on thenucleotide genome sequence of ITS and the chloroplast gene of rbcL. Then, the effects of differentNaCl concentrations on the physiological and biochemical composition, the activities andtranscription pattern of several carbon and nitrogen metabolism enzymes of the alga werecompared.The aim of this paper was to confirm its species, toscreen the optimal salinityconditionsof this alga, and to explore the inorganic carbon and nitrogen enzymes to the salinity, which wouldprovide data for the salt tolerant mechanisms.Dunaliella sp. D3was determined its taxonomy based on the nucleotide sequence of ITS andthe chloroplast gene of rbcL. The distances ranged from0.026to0.136based on the ITS sequenceof D3and the other23strains of Dunaliella, and the distance varied from0.005to0.060based onthe rbcL sequence of D3and the other21strains of Dunaliella. The genetic distances andphylogenetic tree confirmed D3as D. viridis.D. viridis D3was used as the experimental materials to study the effect of four kinds of NaClconcentrations on the growth, chlorophyll content, protein content, total lipid content andchlorophyll fluorescence parameters. The effect of different NaCl concentration on the growth ofD. viridis showed that the algae grew fastest in1.00M NaCl medium, and the cell density of0.44M,2.00M and3.00M NaCl conditions was85.9%,93.2%and80.7%of that of1.00M NaClon the10th day, respectively. The chlorophyll content was the highest in the1.00M NaCl medium.The soluble protein content was the highest in the1.00M NaCl group. The total lipid content of D.viridis increased with the increasing NaCl concentration from0.44to2.00M, and its maximallipid content was detected in2.00M NaCl group. The chlorophyll fluorescence parameters of PSII(Fv/Fm), actual photochemical efficiency of PSII (ФPSII), photochemical fluorescence quenching(qp) deceased under the high salinity, but non photochemical fluorescence quenching (NPQ)increased in3.00M NaCl culture.The effects of four kinds of NaCl concentrations on two carbon metabolism enzymes ofRubisco and carbonic anhydrase activity and their transcriptional expression of D. viridis werestudied by enzyme assays and the quantitative real-time PCR techniques. The results showed thatthe highest initial and total Rubisco activities were detected in the presence of1.00M NaCl,decreasing to37.33%and26.39%of those values in the presence of3.00M NaCl, respectively. Atthe transcriptional level, Rubisco large subunit (rbcL) and small subunit (rbcS) attained theirhighest expression in the presence of1.00and2.00M NaCl, respectively. The extracellular carbonic anhydrase (CA) activity increased as the increasing of NaCl concentration from0.44M to2.00M NaCl, but CA activity was inhibited in the presence of3.00M NaCl. However, the mRNAaccumulation of CAwas salt-inducible and attained its highest expressions in the presence of3.00M NaCl.D.viridis D3was used to study the effects of four kinds of NaCl concentrations on nitratereductase activity and its expression. The results showed that the effect of NaCl on the nitratereductase activity was not significant different, but the effect of NaCl on the NR gene expressionwas significantly different. The mRNA accumulation of NR was decreased as the increasing ofNaCl concentration from0.44M to3.00M NaCl, and its mRNA accumulation in1.00M,2.00Mand3.00M NaCl group were0.87times,0.52times and0.33times of that of0.44M NaCl,respectively.In conclusion, the Dunaliella sp. D3was confirmed as D. viridis, and1.00-2.00M NaCl wasthe suitable salinity for the growth of D.viridis.The Rubisco activity and rbcL mRNAaccumulation responses to the salinity stress were similar to the algal growth, which were alldetected the highest in1.00M NaCl group. CA activity and its gene expression were all induced bya certain concentration of NaCl. While, nitrate reductase activity didn’t show asignificantlydifferent corresponding to the salinity, but its gene expression was inhibited by high salinity.