Mutants Screening Of Arthrospira Platensis With High NH₄ HCO₃ Tolerance And Expression Analysis Of CCM Associated Genes

Arthrospira is hailed as the best food in 21st century by FAO, it has been industrialized farming in 80s of last century. It is important for us to get further improvement of the germplasm quality of Arthrospira and obtain the mutant strain with good traits in industrial production. Moreover, having a better understanding of CO₂ concentrating mechanism(CCM) which closely relates to photosynthesis is essential.NH₄HCO₃ can provide carbon and nitrogen sources. And NH₄HCO₃ is a product that CO₂ of the industrial emissions is absorbed by ammonia-wate, the wild use of NH₄HCO₃ can lead us to a low-carbon lifestyle. NH₄⁺ can kill the protozoa in the industrial cultivated A. platensis. But high concentrations of NH₄⁺ will be harmful for the normal growth of the algae. so it is necessary to breed an A. platensis mutant that can be prosperous in high concentration of NH₄HCO₃. NH₄HCO₃ is a product of ammonia fixating CO₂, andIn addition, the mechanism of A. platensis CCM can increase the concentration of CO₂ around Rubisco in the case of low CO₂ concentrations. The mechanism of A. platensis plays an important role in the photosynthetic process. Carboxysome is a main component of CCM. The genes of ccmM and ccmO encode Carboxysome's embedded protein, and CcmM's N-terminal domain is similar toγ-carbonic anhydrase. Therefore, this paper attempts to understand ccmM and ccmO gene expression characteristics of A. platensis by the method of RT-PCR, providing us a better understanding of the mechanism of CCM.In this thesis, in NO3-N-free Zarrouk medium, We measured the growth rate of A. platensis cultured with different concentrations of NH₄HCO₃, verified A. platensis tolerance for NH₄HCO₃. We compared the mutagenic effects of A.p mutagenesised by UV, EMS, NTG and the growth of algae strains at high NH₄HCO₃ concentrations after mutagenesis were also monitored, providing enough evidence to screen suitable NH₄HCO₃–tolerant mutants of A. platensis .The results showed that, in NO3-N-free medium, 0.004mol/l of NH₄HCO₃ could improve the growth of A. platensis while 0.022mol/l of NH₄HCO₃ suppressed its growth and would be lethal to it if the concentration continues to rise. The NH₄HCO₃ tolerance limit of A. platensis is about 0.022mol/l L.The comparison of the algae strains growth showed that in the stress of high NH₄HCO₃ concentrations the growth rate of NTG-treated algae strains varied the most, thus the NTG-treated algae were most likely to yield NH₄HCO₃–tolerant strains.By screening 1212 A. platensis mutants with NTG treatment under 0.022mol/l NH₄HCO₃ stress, 8 NH₄HCO₃-tolerant mutants were obtained, 5 of which embraced a considerably high tolerance to NH₄HCO₃ comparing to the original A. platensis. Through the comparison of the algae strains, the growth rate of No.2 mutant strain was 29.3% improved than the original alga when the NH₄⁺ concentration is 0.004mol/l. The growth rate of No.5, 6, 8 mutant strains were positive values while the original strain was negative value when the NH₄⁺ concentration was 0.022mol/l. The result indicated that at that stress condition, the mutant strains can survive while the original strain can not. The maximum net photosynthetic rate of No.2 mutant strain was 3.066×10^-4nmol·h^-1·cell^-1 when the NH₄⁺ concentration was 80mg/L,14.8% improved than the original alga. The maximum net photosynthetic rate of No.4, 5, 6, 8 mutant strains were 2.883×10^-4nmol·h^-1·cell^-1, 3.027×10^-4nmol·h^-1·cell^-1, 2.969×10^-4nmol·h^-1·cell^-1, 3.310×10^-4nmol·h^-1·cell^-1 respectively when the NH₄⁺ concentration was 160mg/L, 91.4%, 101.00%, 97.1%, 119.79% improved than the original alga. The genetic stability were verified using a growth curve test.On the other hand, A. platensis was cultured with 2% CO₂ 48hrs to inhibit the expression of CCM-related inducible genes, then passed into the air containing low concentrations of CO₂ to induce the expression of CCM-related inducible genes. After induced by low-carbon, changes of A. platensis photosynthetic efficiency was measured. By the method of RT-PCR, the expression of ccmM, ccmO related to CCM is studied after the induction of low-carbon.The results show that the photosynthetic efficiency of A. platensis was decreased. Photosynthetic efficiency was stable about 30min after the low-carbon induction. the expression of ccmM and ccmO did not change significantly after the low-carbon induction.. It shows that the number of Carboxysome do not change significantly after a short low-carbon period of induction. And the protein encoded by ccmM can not play the role of carbonic anhydrase. There may exists another carbonic anhydrase in the algae.In summary, this thesis shows that inducing and screening of enhanced mutant stains can be an effient method in A. platensis breeding. And we can prove that the number of Carboxysome do not change significantly after a short low-carbon period of induction, the protein encoded by ccmM can not play the role of carbonic anhydrase.