Research On Knockout Of NADPH Oxidase Gene In Rice And Tomato To Create Crop Male Sterile Line

Crop hybrids have significant heterosis compared with their inbred parents,can significantly improve crop yield,disease resistance and stress resistance,and have important commercial value.Generally,the preparation of crop hybrids requires the creation of plant male sterile lines,and the use of plant male sterile lines can effectively reduce breeding costs.However,the selection and transfermation of sterile lines usually requires a long breeding cycle,which limits the breeding and popularization of sterile line varieties.Using CRISPR/Cas9 gene editing technology to knock out key genes in the development of plant anthers,male sterile lines can be directly obtained,significantly shortening the breeding period of creating sterile lines,and greatly improving the process of hybrid seed production,which has important economic value.In this study,two model plants of monocotyledonous rice and dicotyledonous tomato were selected as experimental materials.Using CRISPR/Cas9 technology to edit specific genes and create male sterile lines controlled by nuclear genes can provide a reference for artificially creating male sterile lines of other crops(food and vegetables),which has important theoretical and application value.The main experimental results obtained in this research are as follows:(1)This study used CRISPR/Cas9 technology to knock out the respiratory burst peroxidase genes Os RBOHA(Os01g53294)and Os RBOHE(Os01g61880),which were expressed in high amounts during rice anther development,and obtained E1-doublegene-mutant osrboha/osrbohe rice without T-DNA insertion.The results showed that Os RBOHA and Os RBOHE gene loss-of-function mutant plants had no difference in growth and seed setting rate from those of wild type.However,further experiments found that the pollen fertility of the double mutant osrboha/osrbohe was significantly reduced,and the content of ROS in anthers was significantly reduced.About 50% of the pollen grains cannot accumulate organic matter,leading to abnormal development of some pollen grains and forming semi-male sterile plants.(2)This study used CRISPR/Cas9 technology to knock out the respiratory burst peroxidase genes Le RBOH(Solyc01g099620)and Le RBOHE(Solyc07g042460)that were highly expressed during tomato anther development.We obtained the tomato single-gene mutant lerboh,lerbohe and double gene mutant lerboh/lerbohe.The results showed that the pollen fertility of the Le RBOH gene deletion mutant plant was normal,and there was no difference between the normal plant and the wild type.the Le RBOHE gene loss-of-function mutation and the simultaneous loss-of-function mutations of the Le RBOH and Le RBOHE genes would cause tomato The pollen is aborted,but the pistil of the double mutant is not affected,and can accept wild-type pollen to bear fruit normally,and the tomato mutant shows male sterility.Further research found that the content of ROS in anthers of lerbohe-1 single mutant was reduced,and starch accumulation was insufficient during pollen development,which led to partial abortion of pollen,but there were still a small amount of pollen that could develop normally.In the lerboh/lerbohe-1 double mutant,the content of ROS in tomato anthers was significantly reduced,and the tapetum PCD was delayed during the development of the anthers,which prevented the pollen from accumulating organic matter,and eventually caused the pollen to abort completely and fail to complete fertilization.The plants exhibited male sterility characteristics.Therefore,we speculate that Le RBOH and Le RBOHE may have overlapping functions in regulating the development of anthers,but the latter plays a major role and the former plays a secondary role.Therefore,we believe that knocking out the Le RBOHE gene alone can obtain a tomato male sterile line,but further knocking out Le RBOH can make the male sterility characteristics more sufficient and stable.In this study,a functional loss-of-function mutant of tomato RBOH gene was obtained by using CRISPR/Cas9 technology,and a male sterile line controlled by tomato nuclear gene was created,which has important commercial value for tomato hybrid seed production.