Screening Of Salt-tolerant Algaes With High Bioactivity And Studying On The Genetic Engineering Cyanobacteria For Degrading Microcystins

In this paper, the samples collected from several regions were screened for salt-tolerant algae, and then the experiment was conducted to study their antibacterial activity and antioxidant activity. The following results are obtained:1. 40 strains of salt-tolerant algae(1% and 3% salinity), as well as 4 strains resistant to 8% salinity were screened and have been conservated; the growth curves of No. 4-9 salt-tolerant algae were measured at 0% and 3%salinity respectively to make sure a better salinity. Besides, the growth curves of 4 strains(8% salinity) were measured, and all of them were identified as Dunaliella salina. The phylogenetic trees were constructed based on ITS sequence analysis.2. Study on antibacterial activity of No. 1-16 algae were performed.Among them, the ethanol extract of No.9 showed a best antibacterial activity to E. coli and Staphylococcus aureus (Diameter of inhibition zone was 14.0 mm and 12.0 mm respectively); the ethanol extract of No. 16 can preferably inhibit the growth of Fusarium moniliforme, Alternaria alternata,Ascosphaera apis(Relative inhibition rate was 7.84%, 14.81%, 14.52%), and the ethanol extract of No. 11 can inhibit Fusarfum tricinctum better( 10.64%).Fatty acids was determined in the strains with better antibacterial activity. It was found that the proportion of unsaturated fatty acids was mainly concentrated in linoleic acid, linolenic acid and hexadecenoic acid or hexadecadienoic acid.3. Study on antioxidant activity of No. 1-16 algae were conducted,including the total phenolics(TP), total flavonoids(TF) and DPPH/ABTS free radical-scavenging activity. The results showed that the methanol extract of No.16 contained the most TP(8.74 mg GAE (g d.wt)-1) and the ethanol extract of No. 16 showed the most TF(19.22 mg QE�(g d.wt)-1); the methanol extract of No. 10 showed the highest DPPH scavenging activity(78.23%) and the methanol extract of No.16 had the highest ABTS scavenging activity(93.84%).Besides, we successfully constructed a genetic engineering strain 6803-A+ used for the degradation of microcystins. The following results are obtained:1. The Mlr A enzyme activity was detected in the cell lysate(0.108 U�mL-1)of 6803-A+ but not in the medium; the MlrA activity varied from the sonication cycles, and it showed 0.149 U mL'1 when sonicated 15 cycles(about 24.83 times of 3 cycles).2. When OD=1, the maximum MlrA activity was 0.108 U mL-1 and 2.985 U�mL-1 for 6803-A+ and BL21(DE3)-pGEX-mlrA respectively, which had a 27.64 times. When OD=2, the MlrA activity of the whole viable cells of Sphingomonas sp. and 6803-A+ were measured, which showed 0.366 mU�mL-1, 0.016 mU�mL-1 respectively.The MlrA activity of 6803-A+ was not high as the other strains, which might be related with the promoter, leader peptide gene; but the success of showing MlrA activity did demonstrate the scheme is feasible, and it broadened a new direction of gene engineering for microcystins degrading.