| Rice is one of the most important food crops in the world.More than half of the world’s population uses rice as a staple food,and the proportion in China is as high as 60%.At present,under the situation that the population of our country is constantly increasing and the area of cultivated land is decreasing,increasing the grain output per unit of land area is the only way to ensure our country’s food security.The successful cultivation of hybrid rice in the mid 1970s was a major breakthrough in the history of agricultural development in the world.It was hailed as the "second green revolution"following rice dwarfing and breeding,creating huge economic and social benefits.The success of hybrid rice involves the transmission of male and female gametes,and the male and female gamete formation pathway is a series of complex and conservative processes,which are essential for plant reproductive growth and material inheritance.In flowering plants,the development of male and female gametes must undergo meiosis.However,meiosis is a precise and very important process,and its internal molecular regulatory mechanism still needs further research and elaboration.In this study,a rice male and female sterile mutant phs1 was used as the material,and its phenotype,physiology,genetics and gene function were studied in detail.The specific research results of this paper are summarized as follows:（1）Phenotype identification of phs1 mutant.The male and female sterile mutant phs1 is a stable genetic mutant selected from the mutant library of wild type Ningjing 4 obtained by EMS mutagenesis.It has no significant difference in vegetative growth compared with wild type plants,but in reproductive growth Obviously,the anthers of phs1 became smaller and whiter at this stage.Iodine-potassium iodide staining showed that there were no fertile pollen grains in mature anthers of phs1,and the pollen abortion mode was completely canonical.Scanning electron microscope observations showed that the mutant pollen grains were uneven in size and irregular in shape;transmission electron microscope observations revealed that there was no accumulation of starch grains within the pollen grain wall,and the development of pollen grain walls was incomplete,resulting in the mutant’s pollen being completely aborted..In order to confirm whether the female gamete development is normal,we saturated pollinated the mutant and found that it still failed to set fruit normally,indicating that the mutant embryo sac was also abnormal.Subsequently,we observed the development of the mutant embryo sac using laser confocal scanning electron microscopy and found that it could not perform normal meiosis to form a tetrad,which resulted in loss of functional macrospore differentiation,and no seven-cell eight-nucleus normal embryo sac formed.The embryos that caused the mutants also aborted.Later,through cytological experiments such as magenta acetate staining and semithin sectioning,it was determined that the pollen abortion period of the phs1 mutant occurred between the period of microspore mother cells and the period of meiosis.This indicates that the developmental defects of phs1 pollen may be related to abnormal meiosis.In order to further investigate which meiosis process is a problem,we used DAPI staining to observe its chromosomes.We found that the phs1 mutant could not normally complete the association of homologous chromosomes in the early stage of meiosis.Pairing,so that they cannot form unequal division of divalent bodies and chromosomes,which eventually leads to their inability to form normal male gametes.（2）Map-based cloning and functional analysis of the phs1 mutant.We used Ningjing 4 and N22 to assemble,enough to construct the F2 population for target gene mapping.Subsequently,we selected 224 extreme plants from a population of 3000 plants.These plants had the same phenotype as that of the parent of Ningjing 4 mutant,that is,closed glume and complete spikelet sterility.We extracted the DNA from the leaves of these 224 extreme individuals and used PCR and polypropylene gel electrophoresis to locate the gene of interest in a 5.7Mb interval near the centromere of chromosome 6.Then we screened and analyzed the candidate genes in the interval.We found a gene called PHS1（POOR HOMOLOGOUS SYNA PSIS1）,which occurred at the junction of the third exon and the intron.The G to A mutation caused its introns not to cut normally,and eventually caused the gene to lose function.（3）Knockout verification of candidate OsPHS1.In order to further determine that the candidate gene OsPHS1 is the target gene we are looking for,we used crispr-cas9 technology to knock out the candidate gene in wildtype Ningjing 4 and finally we obtained 24 knockout plants,and 8 of them were positive Plants,these plants have the same phenotype as the Ningjing 4 mutant,namely closed glaze and complete infertility,and their pollen has also been completely defeated by iodine-potassium iodide staining.Therefore,the candidate gene OsPHS1 is our Find the gene of interest.In this study,we cloned a new meiotic-related gene OsPHS1 in rice,and analyzed the phenotype after mutation,and found that its mutation would cause abnormal expression of gametes during meiosis Type,which ultimately leads to its inability to form fertile gametes.These works will be of great significance for us to further understand the process of meiosis and explore new molecular regulatory networks. |
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