| The carbon source and nitrogen source are components that consist of important proteins and nucleic acids in cyanobacteria.Therefore,the proportion of carbon and nitrogen needs to be maintained at a stable level to maintain the normal life activities of cyanobacteria.In order to maintain the balance between carbon and nitrogen,cyanobacteria develop a variety of mechanisms that regulate the balance between carbon and nitrogen,since the external environment for the growth of cyanobacteria is always changing.Cyanobacterial NDH-1 is located on the thylakoid membrane and mediates respiration,cycle electron transport around the photosystem I(PSI),and carbon dioxide uptake.These three functions mainly foucus on carbon metabolism.P? is a signaling protein widely present in bacteria,archaea,and plants and it plays a key regulatory role in nitrogen metabolism.Accoding to previous studies,we have found that P? dephosphorylates in the ndhB-deletion mutant(M55),suggesting that NDH-1not only regulates carbon metabolism,but also regulates nitrogen metabolism.So,can P? protein regulate carbon metabolism by regulating the activity of NDH-1?In this thesis,the P?-deletion mutant(?P?)was constructed and its effects on the activity of NDH-1 were detected at the physiological level.The effect of?P? on the activity of NDH-1 was verified by constructing double mutants of P? and NDH-1subunits.The main research results were summarized as follows:(1)To test the effect of P? protein on NDH-1 activity,we constructed the P? deletion mutant(?P?)and overexpression strain(OX-P?).By monitoring the post-illumination rise in chlorophyll a fluorescence in WT,?P? and OX-P?,it was found that deletion of P? can increase the total activity of NDH-1,and overexpression of P? can reduce the total activity of NDH-1.(2)To determine the effect of P? on the activity of NDH-1,we detected P700redox kinetics and re-reduction of P700~+in darkness in WT,?P?,and OX-P?.We found that deletion of P? can increase NDH-1-dependent cyclic electron transport(NDH-CET),while overexpression of P? can reduce NDH-CET.By measuring the respiration rate of both WT and?P?,it was found that deletion of P? can increase NDH-1-dependent respiration rate(NDH-Respiration).(3)To confirm the effect of deletion of P? on the activity of NDH-CET,we knocked out the NDH-1 subunit NdhV,which only affects the activity of NDH-CET,in the background of P? deletion mutant,and measured its NDH-CET activity.The result showed that the NDH-CET activity in the P? and ndhV double mutant(?P?/ndhV)was lower than that in?P? single mutant,which indicated that?P? indeed increased the rate of NDH-1-dependent cyclic electron transport.To confirm the effect of P? deletion on NDH-Respiration activity,we knocked out the NDH-1subunit NdhD2,which affects the NDH-Respiration activity,in the background of P? deletion mutant,and measured its NDH-Respiration activity.The result showed that NDH-Respiraton activity in the P? and ndhD2 double mutation(?P?/ndhD2)was higher than that in?P? mutant,indicating that?P? indeed increases the respiratory rate mediated by NDH-1.(4)In order to determine the effects of NDH-1-mediated respiration and cyclic electron transport on the growth of?P? mutant,we determined the levels of ROS in WT,?P?,?P?/ndhV,and?P?/ndhD2.The results showed that the ROS levels in?P?/ndhV were higher than those in WT,whereas the ROS amounts in?P?/ndhD2were lower than those in WT.This suggests that the increased activity of NDH-1 in?P? can reduce ROS content.In summary,the deletion of P? in cyanobacteria increases the activity of NDH-1dependent respiration and cyclic electron transport,and consequently the increase of NDH-1 activity in?P? helps to reduce ROS accumulation in cyanobacterial cells. |
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