Functional Analysis Of Pollen Mitosis Relative Pmr And Pollen Germination Relative Ossta99 In Rice

As the important link of spermatophyte sexual reproduction,male gametophyte development has always been a research focus.The microspore undergoing twice mitosis to form mature pollen grain is the key process of male gametogenesis,and pollen germination and pollen tube elongation are necessary for the double fertilization.However,the molecular mechanisms of that processes have not well elucidated.Here,we focus on the function analysis of PMR(Pollen Mitosis Relative)and pollen germination relative gene OsSTA99 in rice via RNAi and the latest CRISPR technology,to investigate further evidence for the regulation network of rice anther development and pollen germination.The main results are as follows:1.The biological function of PMR in riceThe results of real-time PCR and in situ hybridization showed that PMR preferentially expressed in the microspore of anther,especially at the bicellular stage of pollen development.The pollen fertility was significantly decreased in the CRISPR plants and the knockdown transgenic plants of PMR-RNAi.Cytological analysis of the PMR-RNAi anther showed the abnormity of male gametogenesis that most of microspores delayed the first mitotic division and failed to undergo pollen mitosis II,formed immature bicellular pollen grains at the anthesis,and that defective phenotype was confirmed by the DAPI staining,eventually resulted in the serious decrease of seed-setting rate.The expression analysis of relative genes in the PMR-RNAi revealed that those genes participating in the middle-late stage of pollen development like ADF,API5 and AIP1 decreased consistent with PMR,that confirmed the period this gene function in.The general consistency of PMR and CDKB2;1 expression tendency also suggested the particular link between them.Clustering and GO annotation analysis among PMR and its co-expressing genes indicated that they were liable to participate in pollen development involving DNA metabolism in nucleus,cell cycle regulation and reproductive structure development.Moreover,the three domains of PMR protein PHD,SWIB and Plus-3 might share common functions in the whole regulation of pollen development.2.The biological function of OsSTA99 in riceThe expression analysis via real-time PCR and in situ hybridization showed that OsSTA99 preferentially expressed at the mature stage of pollen development.In vitro pollen germination and in situ staining of pollen tube of the OsSTA99-RNAi showed that most of pollens could not germinate normally and the shape of new pollen tubes was unusual,ultimately severely damaged the fertilization and seed setting.There were not any obvious visual phenotypical abnormalities observed in the outer wall of anther,pollen exine or the germinal aperture using SEM(scanning electron microscopy).The results of fluorescence labeled staining in OsSTA99-RNAi observed that the structure of microfilament was abnormal,and the pollen tubes were short and distort,showing variable in direction.Expression detection of relative genes in OsSTA99-RNAi revealed that the GEM encoding microtubule-binding protein and the sucrose transporter SUT1 both had great correlation with OsSTA99,implying that the regulation mechanism of it may involve the cytoskeletonic system and material transport.In addition,the protein of OsSTA99 contained the Aptl domain,may relate to the vesicle trafficking in the pollen tube tip.