The Effect Of Cytoplasmic Male Sterility Gene Orf182 On Mitochondrial Respiratory Chain Complexes Of D1-CMS Rice

Cytoplasmic male sterility(CMS)is not only the basis of crop hybridization utilization,but also an ideal material for information communication between nucleus and cytoplasm.At present,many types of cytoplasmic male sterility have been reported in rice,among which only a few have been widely used in agriculture.Therefore,it is very important to explore new cytoplasmic male sterility types and study their mechanisms for the development of crop breeding.D1-CMS rice is a new type of sporophyte CMS rice,which is independent of the Wild Abortive rice and shows non-pollen-type abortion,developed from dongxiang wild rice in Jiangxi province.In this study,combining with the analysis of mitochondrial targeted metabolomics,we attempt to reveal the molecular mechanism of D1-CMSorf182 using mitochondrial system,anther cells and prokaryotic expression system.The main results are as follows:The DPA sterile lines and maintainer DPB anther development in different periods of mitochondrial metabolite content are analyzed by mitochondrial targeting metabolomics,the results showed that 24 kinds of metabolites have differences in the process of anther development in the DPA and DPB,and the citric acid cycle pathway involved in energy metabolism may be related to DPA anther abortion.Differential centrifugation and Percoll gradient centrifugation were used to extract purified mitochondria from the etiolated rice seedlings of sterile line DPA and maintainer line DPB.The mitochondrial physiological and biochemical indexes of sterile lines and maintainers were measured.The results showed that the mitochondrial membrane potential and ATP content in sterile lines decreased significantly compared with maintainers.The subcomponent structure of mitochondria was separated and Western blotting analysis was further performed using ORF182 monoclonal antibody.The results showed that ORF182 was distributed in the mitochondrial Mitoplast(the complete structure of mitochondrial inner membrane and stroma).BN-PAGE was used to successfully separate DPA and DPB mitochondrial respiratory chain complexes under native conditions,and Western blotting revealed ORF182 signals in mitochondrial respiratory chain complexes.Histochemical enzyme activity staining was used to compare and analyze the mitochondrial complex activities in DPA and DPB.Different activities of both mitochondrial respiratory chain complex Ⅰ and complex Ⅴ were detected between DPA and DPB,while no significant difference of mitochondrial complex Ⅱand complex Ⅳ activities were found.Our previous studies on paraffin sections of anther cells showed that the anther development of DPA and DPB began to differentiate at the meiosis stage of microspore mother cells,probably caused by abnormal disintegration of anther tapetum cells.In this study,TUNEL was used to detect the apoptotic characteristics of anther tapetal cells in DPA and DPB at different stages of anther development.Preliminary results showed that the anther abortion of type D1 rice may be related to the delayed disintegration of anther tapetal cells.Previous studies have shown that the expression of ORF182 had a significant inhibitory effect on the growth of E.coli,and lead to abnormal outbreak of ROS content and significant decrease of ATP content in E.coli.However,the corresponding molecular mechanism of the effect is unclear.We used q RT-PCR to detect the expression levels of anaerobic sensitive marker genes Arc A,Arc B and Fnr in p ET-28a-orf182 expressing strain and its control strain under aerobic and anaerobic conditions respectively.The results showed that,the expression levels of Arc A and Fnr genes in p ET-28a-orf182 were significantly higher than those in the control strain under aerobic condition,while the expression levels of Arc A and Fnr genes were not significantly different between the expression strain and the control strain under anaerobic condition.It suggests that the expression of ORF182 affected the oxygen utilization of E.coli.Further,BN-PAGE technology and Western blot analysis showed that ORF182 was present in the plasma membrane respiratory chain complex of p ET-28a-orf182 expressing strain.In addition,enzyme activity staining results showed that the activity of plasma membrane respiratory chain complex Ⅰ was different between p ET-28a-orf182 expressing strain and p ET-28 a control strain,while the enzyme activity of complex Ⅱ was not significantly different between them.