Selection For The Resistant Mutants Of Spirulina Platensis To Amino Acid Analogues And Studies On Their Biological Characteristics

In this thesis, the effects of ultrasonics on Spirulina platensis A9 strain werestudied, the optimum time parameters of to breakdown the filaments of A9 strainby ultrasonics were obtained. The sensitivity of A9 strain to ρ-fluorophenylalanine(FPA) and L-canavanine sulphate(CS) was investigated. Ultraviolet (UV), ⁶⁰Co-γray and ethyl methane sulphonate (EMS) were used as mutagens. Their effects on thesurvival and mutagenesis of A9 strain were studied, and two stable mutants resistantto amino acid analogues (AAAs) were obtained, and the biologic characteristic of themutants were studied, which can offer valuable markers for further genetic study. Thefollowing results have been obtained. The average length of the fragments of A9 strain was about 1.47 cells after 40s'pretreated by ultrasonics, and these fragments still had a perfect regeneration ability,so it was a suitable materials to mutation study. The A9 strain was sensitive to FPA and CS. The minimum inhibitoryconcentrations (MIC) of FPA and CS in liquid medium were 1μg·ml^-1 and 0.5μg·ml^-1respectivly, while the MICs of FPA and CS both in solid medim were 15μg·ml^-1. FPAand CS were suitable selection genetic markers for the A9 strain. S. platensis A9 strain in log phase was broken by 40s pretreatment of ultrasonics,the sample was mutagenized by ultraviolet (UV),⁶⁰Co –γ ray and ethyl methanesulphonate (EMS) respectively, then the samples were inoculated into liquid and solidZarrouk Medium after 3h pre-incubation. With the increase dosage of mutagens thesurvival ratio of samples were decreased dramatically and under the condition of UVfor 7.0 J·m², ⁶⁰Co –γ ray for 2500 Gy and EMS for 1%, no growth occurred onplates, whereas the growth of the samples with high dosage treatment in liquid culturecould somehow resumed. The samples mutagenized were inoculated to liquid mediumwith 20μg·ml^-1 FPA and 20μg·ml^-1 CS, respectively, in a light incubator for 3-4d,then were spread on plate with the same concentration of FPA and CS. The resistantmutation rates were calculated after growing for about 30d. The lethal dosage (LD) and the D37 dosage, at which 37% population cansurvival , of UV were 7.0 J·m² and 2.2 J·m² respectively,and LD/ D₃₇=3.18. Thesurvival of A9 strain was 28.7% with 2.5 J·m² treatment of UV, and the resistantmutation rates to the two amino acid analogues (AAAs) was greatly increased withthe highest resistant mutation rates to FPA and CS being 2.31×10-3 and 1.5×10-3,respectively. The maximum mutagenic effects (MME) of UV for the two AAAs were48.53 and 52.63, respectively. The LD and D37 of ⁶⁰Co-γray were 3500Gy and 1250Gy, and LD/ D₃₇=2.8. Themutation rate of strain A9 were notably increased by ⁶⁰Co-γray at 2500 Gy, at whichthe survival rate was 10.49%, and the highest mutation rate to FPA and CS were 5.07×10-3 and 9.64×10-4 respectively, and the MME of 60Co-γray for the two AAAswere 241.43 and 74.15, respectively. The LD and D37 of EMS were 1% and 0.3% respectively, and LD/ D37=3.33. Thesurvival of A9 strain was 13.2% with 0.4% EMS, and the resistant mutation rates tothe two amino acid analogues (AAAs) was greatly increased with the highest rates toFPA and CS being 4.9×10-4 and 3.24×10-4, respectively. The maximum mutageniceffects (MME) of EMS for the two AAAs were 5.25 and 32.4, respectively. Compared to UV and EMS, the ⁶⁰Co-γray could cause more severe damage toA9 strain (lower LD/ D37 value) and induce stronger MME (higher value). Theresistant mutation rates to FPA induced by the three mutagens were all higher thanthat to CS. Two stable mutants resistant to amino acid analogues were obtained, andtheir biologic characteristics were somehow changed. Compared to wild A9 strain thefilaments pattern of the mutants have changed, their growth ratios were slower, theresistant to the same amino acid analogues were greatly increases, their contents ofchl-a,caroteniod and phycobiliprotein were lower, and corresponding amino acidswere acuumulated.