Cloning And Functional Analysis Of Male Sterility Gene DCET1 In Rice

In Angiosperms,anther development comprises of various complex and interrelated biological processes,critically needed for pollen viability.The timely degeneration of tapetal cells is essential for callose biosynthesis and degradation and pollen wall formation by providing nutrients for microspores development.Here,we identified and functionally characterized a rice male sterility mutant dcet1(defective in callose,exine and tapetum1)from the mutant library induced by EMS mutagenesis under the background of indica rice cv.Zhonghui 8015,and the candidate gene was subsequently cloned and its functions were analyzed.The results are shown as follows:1.Compared with wild-type plants,dcet1 mutant displayed normal vegetative growth,but the anthers of dcet1 mutant were weaker,smaller and whitish yellow.After staining with I2-KI,the dcet1 mutant was found completely sterile with whitish yellow and ill-shaped pollen grains.Cytological observations showed that the microsporocytes in dcet1 displayed abnormal callose and pollen exine as well as delayed tapetum degeneration.2.The F2 population was generated from a cross between dcet1 and Zh8015 for genetic analysis and the results showed dcet1 male sterility phenotype was controlled by a single recessive nuclear gene.Then a F2 segregation population obtained by crossing the dcet1 mutant and 02428(japonica),the DCET1 locus was delimited to an 84 kb region between the markers Indel8 and RM43 on chromosome 8.Subsequently sequencing analysis revealed a single nucleotide polymorphism(SNP)substitution from G(Threonine-192)to A(Isoleucine-192)located at the fifth exon of LOC_Os08g02330 in dcet1 mutant.Functional complementary and CRISPR/Cas9 system knockout results confirmed that DCET1 is the gene responsible for the dcet1 abnormal callose,defective pollen wall formation and delayed tapetum degeneration.Phylogenetic analysis shows that DCET1 belongs to RNA recognition motif(RRM)containing family also called as RNA-binding domain(RBD)proteins.3.In the chemical analysis of anther cuticular lipids,the dcet1 displayed a significant reduction in the anther cutin monomers,while the wax constituents were much higher than the wild-type.q RT-PCR showed the DCET1 expresses in root,stem,leaf and sheath,but predominant expression in anthers at stage 7 and 8.GUS analysis was found to be consistent with q RT-PCR,showing the highest relative expression levels in anthers at stage7 to 8.In summary,the current study presents a novel RNA recognition motifs(RRM)-containing protein,named as DCET1(Defective in Callose,Exine and Tapetum1)in rice.Briefly,the DCET1 regulates callose biosynthesis and degradation,pollen exine formation by affecting exine wall patterning,including abnormal nexine,collapsed bacula,irregular tectum,and timely PCD by delaying the tapetal cells degeneration.Taken together,disruption of DCET1 function leads to the extension of male sterility and reproductive system in rice and may play an important role in hybrid rice breeding programs.Also,being the closest homolog of Arabidopsis CID-like proteins of mainly unknown functions,DCET1 receives key importance for predicting and characterizing RBDs in general and CIDs in specific in all eukaryotes.This study will further strengthen our knowledge to understand the molecular mechanism of rice fertility and will provide theoretical basis for the use of heterosis.