Map-based Cloning And Functional Analysis Of Male Sterility Gene OsFIGNL1 In Rice

Rice is an important crop,and serves as a staple food for half of the world's population.Research into the mechanisms of male sterility contributed to resolving production problems.Male sterility has a significant impact on rice yield,however,it also provides convenience for genetic improvement of rice hybrids.The research on male sterility mutants helps in understanding molecular mechanisms of male sterility.Male reproductive development is a complex process composing many biological events and highly ordered developmental process.Therefore,understanding the molecular mechanism of male sterility plays an important role in rice breeding.In this study,mutagenesis was performed by treatment with ⁶⁰Co-?ray of Zhonghui8015,resulting in identification of the complete male sterility mutant fignl1.In order to explore the reason of male male sterility,phenotypic differences between the anther of the wild type and the fignl1 mutant were analyzed by observations of the anther transverse sections,SEM,TEM and PMC staining.In addition,studies on map-based cloning,transgenic experiment,and functional analysis were carried out.The main results showed as follows:1.Through the morphological and cytological observation of rice male sterility fignl1,we observed that there was no obvious difference between the wild type and mutant plants in the vegetative stage,however,the mutant could not set seeds.Compared to wild-type anthers,the mutant anthers were relatively small,thin,and slightly yellow.The mutant successfully set seeds when the fignl1 mutant plants were pollinated with wild-type pollen grains,indicating that female fertility was not severely affected in the fignl1 mutant.Based on the SEM observation,mature pollen grains of fignl1 pollen grains were irregular,shrunken,and failed to produce internal contents and nuclei.The pollen intine and extine of the fignl1 mutant had an abnormal morphology,and the fignl1 pollen showed a thicker foot layer and tectum.The wild-type anther epidermis was less compact compared to the fignl1 mutant.2.The characterization of cross sections of fignl1 anthers during the various developmental stages demonstrated that tapetum of fignl1 mutants had several abnormalities,including abnormal morphology,a reduced rate of degradation compared with wild type after the vacuolated pollen stage.At the vacuolated pollen,microspores began to form with an abnormal shape and no vacuoles.At the mature pollen stage in the fignl1 anther,the cells of the endothecium appeared expanded,the tapetum remnants persisted with no cell contents,and microspores degenerated with an irregular shrunken shape.In fignl1 mutant meiocytes,chromosomes appearing as single threads were observed at the pachytene stage.During diakinesis,irregularly-shaped chromosomes,chromosome bridges,and entangled mass of chromosomes were visualized.During the second meiotic division,lagging chromosomes and fragments were still observed in the cell,and resulted in formation of the tetrads with nuclei of different sizes from wild type.These results strongly indicated that the loss of function of OsFIGNL1 adversely affected chromosome behavior during meiosis,finally lead to defects of microspore development.3.The genetic analysis revealed that the sterility phenotype was caused by a single recessive mutation.We generated an F₂ population by crossing the fignl1 mutant and Zhonghua11?japonica rice?for map-based cloning of target gene.To finely map the Os FIGNL1 gene,2300 F₂ population individuals exhibiting the fignl1 mutant phenotype were used for genetic analysis.Finally,we mapped the candidate gene to a 250 kb region on chromosome 12 between the newly developed molecular markers S7 and S8,which contained twelve predicted open reading frames,annotated by The Rice Annotation Project Database?RAP-DB http://rapdb.dna.affrc.go.jp/?.Sequence analysis showed that the fignl1 mutant carries a single nucleotide Adenine deletion in the third exon of annotated gene Os12g0443800.1,creating a premature stop codon at the 187 th amino acid residue.In addition,we found that Os12g0443800.2 and Os12g0443800.1 were annotated as a P-loop containing nucleoside triphosphate hydrolase superfamily protein with putative,overlapping transcribed sequences,suggesting that these ORFs may be a single,transcriptional unit.RT-PCR showed that Os12g0443800 containing Os12g0443800.1 and Os12g0443800.2 represented a single mRNA,and the sequence of Os12g0443800.2revealed a mis-annotation in the RAP-DB database?http://rapdb.dna.affrc.go.jp/?.Os12g0443800 consisted of thirteen exons and twelve introns,comprising a 2,085-bp cDNA region,encoding a predicted protein of 694 amino acids.Functional complementary experiments verified that wild-type Os12g0443800 can rescue the male sterility phenotype of the fignl1 mutant plants.4.We examine developmental expression patterns of OsFIGNL1 by qRT-PCR technology.Analysis revealed that OsFIGNL1 was expressed in several tissue,including root,shoot,leaf,sheath,and anther at different developmental stages.Particularly,OsFIGNL1 expressed predominantly in anthers at the meiosis stage,and progressively reduced as anther development proceeded.The subcellular localization of OsFIGNL1 protein showed that OsFIGNL1-green fluorescent protein was exclusively localized in the nucleus in rice protoplast cells.We expressed the AAA domain of rice OsFIGNL1 in Escherichia coli,and found that the rice OsFIGNL1 had highly ATPase activity in vitro.Alignment of these protein sequences and evolutionary tree revealed that FIGNL1 exist widely in monocots,dicots and animal.These results imply that FIGNL1 is a conserved gene in species.5.Yeast two-hybrid and BiFC expriments demonstrated that rice OsFIGNL1 could interact with RAD51A1,RAD51A2,DMC1 A and DMC1 B in vivo.The subcellular localization of RAD51A1 and RAD51A2 in rice protoplast is nuclear.These results provide an in-depth understanding of OsFIGNL1 involving in the molecular mehanisms of homologous recombination during meiosis.In conclusion,our results clearly demonstrate that OsFIGNL1 participates in control of male fertility in rice,and male sterility phenotype of fignl1 was caused by mutation of OsFIGNL1,leading to abnormalities in chromosome behavior in meiosis.