Construction Of Rice OsCatB Knock-out Mutants And Preliminary Analysis Of Their Stress Tolerance

Rice is one of the most important economic food crops,and more than half of the people in the world use rice as a staple food.In recent years,salinization of land,soil erosion and shortage of fresh water resources occurred frequently,which seriously threaten the rice producti on.Therefore,how to improve the stress resistance of rice so that it can grow and develop normally under environmental stress and achieve high or stable yield has become an important issue to be solved urgently.Previously,we have confirmed that rice Cat C plays an important role in the response to salt stress,but the function of Cat B,another member of rice catalase（CAT）family,in the response to stress is not fully understood.It is necessary to analyze the function of Cat B in the stress of adversity for deeply studying the function of CAT family in different stresses.To this end,we constructed the Os Cat B loss-of-function mutants,and carried out a series of studies on the function of Cat B in adversity stress.The detailed results are as follows:（1）In order to determine the expression pattern of Os Cat B under stress,the trend of transcription levels of Os Cat B in rice under salt,high temperature and oxidative stress were analyzed by the quantitative PCR.The expression levels of Os Cat Bwere found to be up-regulated under these abiotic stresses,indicating that Os Cat Bmay play a certain role under various kinds of stresses.Phylogenetic tree analysis showed that Cat B and Cat C are closely related and far away from Cat A,and Mainly based on the activity of catalase.（2）In order to explore the function of rice Cat B in response to stress,we used CRISPR/Cas9 gene-editing system to conduct site-directed editing of Os Cat B of rice variety Kitaake,and a total of 52 independent transformed seedlings were obtained.After subsequent sequencing identification,3 homozygous mutants with different mutation sites were obtained,namely catb-1,catb-2 and catb-3,and the catb-1 and catb-3 were selected as the subsequent research materials.（3）In order to analyze the function of Cat B in a high-salt environment,the mutants catb-1 and catb-3 as well as wild-type（WT）seedlings were treated with salt stress（140 mmol/L Na Cl）for 8 days,and then recovered for 20 days.It was found that there was no significant difference in the phenotypes and survival rates between catb mutants and WT.The physiological changes were further measured,and no significant difference was found in CAT enzyme activity and H₂O₂ accumulation under salt stress.We preliminarily speculate that Cat B dose not play a major role in the response of rice to salt stress.（4）In order to explore the role of Cat B in high temperature and oxidative stress,the phenotypes,CAT enzyme activities and H₂O₂ contents of WT and catb mutants seedlings under high temperature stress were first analyzed.It was found that,compared with WT,catb mutants showed hypersensitivity to high temperature,and their CAT enzyme activities were significantly reduced,while H₂O₂ contents were significantly increased.Secondly,after the simulated oxidative stress treatment by methyl violet（MV）,the WT seedlings had a longer shoots and a better growth state than catb mutants.These results indicate that Cat B can positively regulate the tolerance of rice to high temperature and oxidative stress.In summary,this study verified the functions of rice Cat B under the salt,high temperature and oxidative stresses,and found that Cat B contributes little to the response to salt stress,but plays an important role in the response to high temperature and oxidative stress.In combination with previous studies which found that Cat C plays an important role in the response to salt stress in rice,we preliminarily revealed the main role played by different members of the CAT family in different stress conditions.These results provide experimental materials and theoretical basis for further exploring the molecular mechanism of CAT family in rice in response to stress.