Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination.Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective ape...Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination.Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture.The mutant gene encodes a novel plasma membrane-localized legume-lectin receptor kinase that we named OsLecRK-S.7.OsLecRK-S.7 was expressed at different levels in all tested tissues and throughout anther development.In vitro kinase assay showed OsLecRK-S.7 capable of autophosporylation.Mutation in s13283(E560K)and mutation of the conserved ATP binding site(K418E)both knocked out the kinase activity.Mass spectrometry showed Thr376,Ser378,Thr386,Thr403,and Thr657 to be the autophosphorylation sites.Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees,but did not abolish the gene function in fertility complementation.oslecrk-s.7 mutant plant overexpressing OsLecRK-S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers,and these phenotypes were abolished by E560K or K418E mutation.The results indicated that OsLecRK-S.7 was a key regulator of pollen development.展开更多
Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant.We attempted to construct this system in r...Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant.We attempted to construct this system in rice by using a late-stage pollen-specific(LSP)promoter driving the expression of maizeα-amylase gene ZM-AA1.To obtain such promoters in rice,we conducted comparative RNA-seq analysis of mature pollen with meiosis anther,and compared this with the transcriptomic data of various tissues in the Rice Expression Database,resulting in 269 candidate LSP genes.Initial test of nine LSP genes showed that only the most active OsLSP3 promoter could drive ZM-AA1 to disrupt pollen.We then analyzed an additional 22 LSP genes and found 12 genes stronger than OsLSP3 in late-stage anthers.The promoters of OsLSP5 and OsLSP6 showing higher expression than OsLSP3 at stages 11 and 12 could drive ZM-AA1 to inactivate pollen,while the promoter of OsLSP4 showing higher expression at stage 12 only could not drive ZM-AA1 to disrupt pollen,suggesting that strong promoter activity at stage 11 was critical for pollen inactivation.The strong pollen-specific promoters identified in this study provided valuable tools for genetic engineering of rice male sterile system for hybrid rice production.展开更多
基金This work was supported by the Major Program of Guangdong Basic and Applied Research(2019B030302006)National Natural Science Foundation of China(U1901203,31801344,andU1704232)+3 种基金Natural Science Foundation of Guangdong Province(2018B030308008,2017A030313104,and 2018A0303130270)Shenzhen Commission on Innovation and Technology Programs(JCYJ20180507181837997)China Postdoctoral Science Foundation(2019M652938)the Innovation Project of Graduate School of South China Normal Uni-versity(2017LKXM011).
文摘Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination.Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture.The mutant gene encodes a novel plasma membrane-localized legume-lectin receptor kinase that we named OsLecRK-S.7.OsLecRK-S.7 was expressed at different levels in all tested tissues and throughout anther development.In vitro kinase assay showed OsLecRK-S.7 capable of autophosporylation.Mutation in s13283(E560K)and mutation of the conserved ATP binding site(K418E)both knocked out the kinase activity.Mass spectrometry showed Thr376,Ser378,Thr386,Thr403,and Thr657 to be the autophosphorylation sites.Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees,but did not abolish the gene function in fertility complementation.oslecrk-s.7 mutant plant overexpressing OsLecRK-S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers,and these phenotypes were abolished by E560K or K418E mutation.The results indicated that OsLecRK-S.7 was a key regulator of pollen development.
基金This work was supp orted by the Nationa l Key Research and Development Plan Program(2016YFD0100406 and 2016YFD0101801)National Natural Science Foundation of China(U1901203,31901532,and U1704232)+3 种基金Natural Science Foundation of Guangdong Province(2018B030308008)Shenzhen Commission on Innovation and Technology Programs(JCYJ20180507181837997)China Postdoctoral Science Foundation(2019M652938)Natural Science Foundation of Fujian Province(2017J01427)。
文摘Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant.We attempted to construct this system in rice by using a late-stage pollen-specific(LSP)promoter driving the expression of maizeα-amylase gene ZM-AA1.To obtain such promoters in rice,we conducted comparative RNA-seq analysis of mature pollen with meiosis anther,and compared this with the transcriptomic data of various tissues in the Rice Expression Database,resulting in 269 candidate LSP genes.Initial test of nine LSP genes showed that only the most active OsLSP3 promoter could drive ZM-AA1 to disrupt pollen.We then analyzed an additional 22 LSP genes and found 12 genes stronger than OsLSP3 in late-stage anthers.The promoters of OsLSP5 and OsLSP6 showing higher expression than OsLSP3 at stages 11 and 12 could drive ZM-AA1 to inactivate pollen,while the promoter of OsLSP4 showing higher expression at stage 12 only could not drive ZM-AA1 to disrupt pollen,suggesting that strong promoter activity at stage 11 was critical for pollen inactivation.The strong pollen-specific promoters identified in this study provided valuable tools for genetic engineering of rice male sterile system for hybrid rice production.
