Cloning And Functional Analysis Of A Rice Pollen Development Gene,LSP2

Rice?Oryza sativa?is one of the most important food crops in the world,and feeds 50%of the world's population.It is of great significance to continuously increase the yield of rice.The application of hybrid rice breeding,which is based on the efficient use of male-sterile germplasms,has made great contributions to the increase of rice yields for humanity.During the reproductive process of rice,the pollen exine protects male gametes from various environmental stresses and pathogen attacks,promoting pollen germination.Therefore,normal pollen exine construction is required for functional pollen formation.In recent years,progresses have been made in understanding the development of rice pollen,however,its regulatory mechanism is not fully explored.In order to improve the understanding of rice pollen development,here,we obtained a new male-sterile mutant by screening EMS-induced mutant library in the background of indica rice variety 9311.Using the methods of genetics and molecular biology,the causal gene was cloned.The results were listed as follows:1.Characterization of mutant.Compared to which of the wild type?WT?9311,though the mutant exhibited normal vegetative growth and floral morphology,its anthers were smaller and pale-yellow.The I₂-KI staining analysis further revealed that the pollen grains produced by the mutant were less and shrunk,and could not be dyed.Accordingly,the mutant was named lsp2?less and shrunken pollen 2?.Genetic analysis revealed that the male sterility of the mutant was controlled by a single recessive locus.2.Cytological analysis.Using the anther sections we found that,from the stage 8,lsp2 mutant showed a swollen tapetum,which had disordered degradation in the next stages,compared with which of the WT.Further scanning electron microscopy and transmission electron microscopy observation revealed that mutant anthers had abnormal development of Ubisch bodies and pollen wall.3.Gene mapping.Using the MutMap method based on resequencing,we found that a single-nucleotide polymorphism?SNP??G to A?on chromosome 2 is closely linked to the male sterile phenotype.This SNP is located on the fourth exon of the protein encoding gene.Further sequencing analysis confirmed that the SNP is co-segregated with the mutant phenotype,suggesting this gene might be LSP2.4.Candidate gene function verification.To further affirm that the malfunction of LSP2 is blame for the mutant phenotype,we used CRISPR/Cas9 genome editing system to knockout this gene in Nipponbare?a japonica rice cultivar with normal fertility?.We found that all the knockout plants showed male sterility,which could mimic the phenotype of the lsp2 mutant.Importantly,a further co-segregation analysis of the backcross population showed the separation of male-sterile phenotype as lsp2 mutant.These results confirmed that this gene is LSP2,and indicated that LSP2 is important for rice male fertility.5.Expression pattern analysis.The results of quantitative PCR and GUS histochemical staining showed that LSP2 was highly expressed in roots,young spikelets and anthers,and weak expressions of this gene were detected in pistils,stems,leaves and spikelet hulls.Furthermore,the sections of GUS-stained anther showed that LSP2 was mainly expressed in tapetum.6.Subcellular localization of LSP2.Through transient co-expression of LSP2-GFP construct and a membrane-marker br356 in tobacco?Nicotiana benthamiana?leaves,we found that LSP2 may be localized to the membrane.7.Analysis of related gene expression profile.The results of quantitative PCR showed no significant difference in the expression of OsPKS2 and TDR between the lsp2 and the WT.However,the expression levels of CYP704B2,DPW,OsACOS12and OsNP1 in the mutant were significantly lower than those of the WT,suggesting that the pathway,which LSP2 is involved in,might be associated with these genes.