Microevolution Of Carbon Dioxide Concentration Mechanism（CCM）Gene In Microcystis And Its Responses To The Change Of CO₂ Concentration

As the main inorganic carbon source for photosynthesis in algae,CO₂plays an important role in the growth of algae.Cyanobacteria is one of the oldest prokaryotes.Faced with changes of the CO₂concentration in the environment,they have gradually evolved a unique carbon dioxide concentration mechanism（CCM）,giving it an advantage in low CO₂concentration conditions.However,since the industrial revolution,the CO₂concentration in the atmosphere has continued to elevate at an unprecedented rate.Studies have shown that the elevated CO₂concentration in atmospheric will affect the exchange of water and air in lakes,oceans and rivers,causing changes in the water environment,affecting the metabolism of phytoplankton in the water,and then having a profound impact on aquatic ecosystems and biogeochemical cycles.Therefore,the impact of elevated CO₂concentration on phytoplankton has become a widespread concern for scholars and water management departments.Studies have indicated that the elevate in CO₂concentration is beneficial to the growth and photosynthesis of Chlaamydomonas reinhardtii,Chlorella pyrenoidosa and Scenedesmus obliquus,and increased the abundance,floating ability and toxins in cyanobacteria.However,some studies have shown that the proliferation rate reduced in cyanobacteria with the increase of CO₂concentration,the chlorophyll a in cyanobacteria increased firstly but then decreased.However,the research on the increase of CO₂concentration on phytoplankton mainly focuses on marine phytoplankton,but there are few studies on the response of freshwater algae to the elevated atmospheric CO₂ concentration.Water bloom is a harmful natural phenomenon formed in the process of eutrophication in water bodies,and it is also a manifestation of degradation for aquatic ecosystems.In recent years,blooms are increasing in frequency,magnitude and duration globally in water bodies.Therefore,as the CO₂concentration in the external environment continues to elevate,how does cyanobacteria,especially for Microcystis,respond to the elevated CO₂concentration?At the same time,faced with elevated CO₂concentration,how does Microcystis CCM regulate and form blooms?These issues urgently need to be further studied.Based on this,this paper selected Microcystis as the research object,using different primers（ccm R2-F1/sbt A-R1,bic A-R2/nha S3-R,sbt A-F1/sbt B-R,sbt A-F1/sbt B-R,bic A-F1/sbt A-R1 and bic A-F1/bic A-R1,etc.）perform PCR amplification on different systems in CO₂concentration mechanism（CCM）to explore the microevolution in their CCM;Further explore the differences in growth,photosynthesis and enzymatic activity of different CCM genotypes at different CO₂concentration clarified the physiological response of different CCM genotypes in Microcystis to different CO₂concentration;in addition,fluorescence quantitative PCR was used to explore the relationship between different CCM genotypes of competitive response in Microcystis in order to reveal the adaptation mechanism in Microcystis to different CO₂concentration environments.The main findings are as follows:（1）Through different primers to explore the CCM structure in Microcystin,including bicarbonate absorption system,CO₂absorption system and carbonic anhydrase system.The results shown that all Microcystin had CO₂absorption system and carbonic anhydrase system,therefore CCM gene diversity in Microcystin are mainly manifested in the bicarbonate absorption system,and all Microcystin lost complete sbt A fragments and bic A fragments,suggests that all strains have obvious deletion in CCM system.At the same time,all strains could be divided into two CCM genotypes:bic A fragment deletion and sbt A+sbt B deletion,suggesting that CCM in Microcystin may have genetic microevolution to adapt and maintain its competitive advantage at different CO₂concentration conditions in the external environment.（2）By measuring the growth,photosynthesis and enzyme activity parameters of two genotypes in Microcystis（sbt A and bic A）at different CO₂concentration（200 ppm,400 ppm and 800 ppm）.There are differences in the growth status of Microcystis under different CO₂conditions.With the elevated CO₂concentration,the growth,photosynthesis and enzyme activity parameters of the three bic A Microcystis gradually increase,while the three sbt A Microcystis gradually decreased.The results indicated that the bic A Microcystis is more suitable for growth in high CO₂concentration environments,while the sbt A Microcystis grows better under low CO₂concentration.In addition,We explored competitive response of different（CCM）genotypes in Microcystin by fluorescence quantitative PCR,found that the percentage of bic A gene copy number and cell density in bic A genotypes are more than 50%in a week at the high CO₂concentration condition,while the percentage of gene copy number and cell density of sbt A are more than 50%in a week at the low concentration CO₂condition.Results indicated that bic A genotype had more competitive advantage at high CO₂condition,while sbt A had a competitive advantage at the low CO₂concentration condition.（3）In addition,by analyzing the physiological responses in four sbt A Microcystis with biolovolume 15.58 mg/L,1.15 mg/L,0.229 mg/L and 4.85 mg/L at different CO₂concentration,results indicated that larger biovolume had the better growth,photosynthesis and enzyme activity in all treatment groups,while Microcystis with a smaller biovolume had the worse growth,photosynthesis and enzyme activity in all treatment groups.And it was found that the biovolume of No.18 and No.23Microcystis is larger and the biological volume of No.19 and No.Microcystis is smaller through previous measurements,and the growth of No.18 and No.23 Microcystis is better than No.19 and No.20,indicating that the larger biovolume had better growth.In summary,there is microevolution in the carbon dioxide concentration mechanism（CCM）gene in Microcystis,and there were differences in growth,photosynthesis and enzyme activity in different CCM gene microevolution types under different CO₂concentration condition.The sbt A Microcystis is more suitable at high CO₂concentration condition,while sbt A Microcystis grow better under low CO₂condition.