Cloning And Functional Analysis Of A Key Gene OsLAP6 Controlling Male Fertility In Rice

Plant male sterile is not only an important biological event,but also is basis of heterosis utilization.Under the strategic background of the 3rd hybrid rice breeding^[1,2],it will be a research emphasis and hotspot for us to create more new and diffirent rice male sterile mutants,and further elucidate its molecular mechanism and finally make it applied in hybrid rice breeding.In this research,In virture of screening a specific indica 9311 mutant library where all mutans were produced by EMS chemical mutation we obtained a male sterile mutant—named oslap6.Subsequently,in virture of a series of relevant experimental means and technology,including genetics,molecular biology and cell biology etc,we cloned a key gene that is responsible for mutant oslap6,and further elucidate its potential molecular mechanism.The results could be listed as follows:1.Mutation screening.The initial phenotypic identification of a male sterility mutant oslap6 obtained by screening the rice mutant library found that the anther development was obviously abnormal,and it could not produce fertile pollen grains,which belonged to a kind of typical abortion type.Further genetic analysis ensure that oslap6 is controlled by a nonphotosensitive recessive nuclear gene.2.Relevant cytological analysis.The results of anther transverse section experiment show that,comparing with the WT?9311?,the mutants exposed its defects accompanied by shrinking microspores and degradation-delayed tapetum in the 9th anther development,and eventually developed into vacant and collapsed pollen grains clingy mutually.Then,the observation of ultrastructure by both transmission electron microscopy?TEM?and scanning electron microscope?SEM?further revealed that the mutant oslap6 had the collapsed bacula in the 13rd anther development as well as the less-organization ubisch body with abnormal form in the 12rd anther developement.This indicates that those appeared defective pollen grains in the mutant oslap6 is due to the deformed extine.3.Gene cloning.Combining MutMap technology and co-segregation experiment,we located in a candidate gene?LOC_Os10g34360?closely with a specific SNP loci,which results from a simple base change?G to A?of the second exons of the candidate gene,and consequently causing only a change within its original triplet coding.Further analysis showed that the candidate gene is homologous with PKSA/LAP6^[3]in arabidopsis thaliana-it encodes a polyketosynthase,which is specifically expressed in the tapetum of anther and participates in the biological synthesis of extine sporopollenin.Therefore,we identified LOC_Os10g34360 as a candidate gene and named it OsLAP6/OsPKS1^[5].4.Reverse genetic validation.By using the CRISPR/Cas9 gene editing techniques and some renlevant genetic experiments,we had successfully created some ideal knock-out transgenic japonica where had unfunctional OsLAP6/OsPKS1,and those knock-out mutants had the same infertility defects with mutant oslap6.This indicates that OsLAP6/OsPKS1 is indeed a key gene controlling male reproductive development in japonica and indica.5.Gene expression pattern analysis.The results of both qRT-PCR and GUS tissue chemical staining showed that OsLAP6/OsPKS1 was highly expressed in young panicles and anthers,low in pistil,and hardly expressed in roots,stems,leaves,and glume.Besides,in situ hybridization and protein localization experiment results further showed that OsLAP6/OsPKS1 was only expressed in the anther tapetum of japonica6.Subcellular localization.Tobacco instantaneous expression experiment shows that OsLAP6/OsPKS1 was located on the endoplasmic resection?ER?.7.Protein functional and conservative analysis.By using homologous protein sequence alignment and evolutionary analysis,it was found that OsLAP6/OsPKS1was conservative in lant plants.In summary,we cloned a key gene controlling male fertility in japonica and indica—OsLAP6/OsPKS1,which encodes a polyketosynthase,and is a switch to regulate the male reproductive development by participating in the biosynthesis of the conserved sporopollenin precursors of extine.