Study On Functional Mechanism Of Deletion Of FT1 Reducing The Poison Of NaHSO₃ And Promoting Hydrogen Production In Synechocystis Sp.strain PCC 6803

The hydrogen photoproduction in microalgae is one of the important ways to solve the energy shortage and environmental problem.The production of hydrogen in microalgae is mainly catalyzed by hydrogenases using the reaction that electrons generated by photosystem II?PSII?reduce hydrogen protons to produce hydrogen.However,hydrogenase is very sensitive to oxygen,and thus hydrogenase is easily inactivated under aerobic conditions,thereby limiting the hydrogen production of microalgae.To remove this limitation,some ways are developed to reduce the oxygen content and promote the hydrogen production in microalgae;for examples sulfur-free culture,NaHSO₃ addition,etc.Although all these methods obviously promote the hydrogen photoproduction in microalgae,they impair the activity of PSII and eventually lead to cell death,which limits the electron source of hydrogen production by microalgae,and consequently hydrogen photoproduction cannot be sustained.Therefore,under the anaerobic background,how to maintain high PSII activity and ensure adequate electron sources have become one of the focus and hotspot of research on hydrogen photoproduction in microalgae.Synechocystis 6803 is a unicellular cyanobacterium with a clear biological background and simple genetic manipulation.The Ni-Fe bidirectional hydrogenase localized in the thylakoid membrane can produce hydrogen via the reaction that electrons and hydrogen protons.Therefore,in this thesis,we tried to obtain the high-PSII activity and high-yield hydrogen mutants under the background of NaHSO₃ by screening the Synechocystis 6803 transposon mutant library,and established a simple and effective screening system for mutants with high hydrogen production.We analyzed the mechanism on how the high-producing hydrogen mutant?ft1 promotes hydrogen photoproduction.This thesis provides an important theoretical basis and reference for the genetic transformation of high-yield hydrogen-producing algae such as Chlamydomonas reinhardtii.The main results are as follows:?1?A large-scale analysis of Imaging-PAM was performed to compare changes in Fv/Fm before and after addition of NaHSO₃ by using Synechocystis 6803 transposon mutant library,and two high PSII active mutants were isolated.Using reverse molecular biological methods such as reverse PCR,the insertion of the same gene ft1 in transposons was identified in these two mutants.Further,the?ft1 was determined to be a highly productive mutant of hydrogen by detecting the amount of hydrogen levels accumulated in the?ft1 mutant.Based on the above results,we successfully established a screening system for high-producing hydrogen mutants of Synechocystis 6803 in the NaHSO₃ background,and obtained a high-producing hydrogen mutant?ft1.?2?FT1 encodes an iron transport-associated protein in Synechocystis 6803.The measurements of?ft1 growth,iron content,and accumulation of reactive oxygen species of different species showed that the absence of ft1 reduced the absorption of intracellular iron,slowed the Fenton reaction caused by the reducing agent NaHSO₃,and reduced intracellular hydroxyl radicals accumulation and effectively reduced the accumulation of ROS,thereby slowing down the damage to PSII and promoting the photosynthesis of Synechocystis 6803.