Functional Analysis Of Arabidopsis FACT And DYI1 In Anther Development

In agricultural production,the use of heterosis in cross-breeding can obtain offspring that are superior to their parents in traits such as growth rate and yield.Exploring the relevant molecular mechanism of male sterility regulation process can explore new gene loci and provide theoretical basis for cultivating excellent varieties.In this study,on the one hand,we explored the molecular mechanism of the FACT complex on fertility regulation during Arabidopsis development;on the other hand,we obtained a new Arabidopsis male sterile mutant by EMS mutagenesis,Related studies were carried out to provide more genetic materials for the mechanism research process of anther development.The main findings are as follows:1.Facilitating chromatin transcription complex(FACT),composed of two protein subunits,SSRP1(structure-specific recognition protein)and SPT16(repressor),related studies have shown that FACT can affect nutrition during Arabidopsis development developmental stages of growth and reproductive growth,but the relevant mechanisms by which FACT regulates fertility remain unclear.Therefore,in order to deeply study the mechanism of FACT regulation of fertility,we purchased T-DNA insertion lines of mutants SALK?011283(ssrp-1-2)and SAIL?115?G02(spt16-2)with deletion of their respective subunits of FACT.Preliminary analysis showed that the fertility of the two mutants decreased.Further reciprocal cross experiments proved that the main reason for the decreased fertility in the two mutants was abnormal male gamete development.Genetic analysis showed that the sterility of ssrp-1-2 and spt16-2 mutants was caused by recessive mutations in their respective genes SSRP1 and SPT16.The results of Alexander staining and seed setting rate statistics showed that the fertility of ssrp-1-2 and spt16-2 mutants was significantly decreased,and the ssrp-1-2/spt16-2 double mutant was completely sterile.Statistical results of inflorescence morphological observation showed that the number of stamens in double processes of ssrp-1-2,spt16-2 and ssrp-1-2/spt16-2 was missing,and the degree of stamen loss was more serious in double mutants.Further anther semi-thin sectioning results revealed abnormal cytoarchitecture development in the mutants:ssrp-1-2 and spt16-2 cell layers were disordered in early anther development,and the ssrp-1-2/spt16-2 double mutant had The lack of structural integrity of cells in all layers of the medicine chamber is more serious.The disordered development of cells in each layer of the drug chamber led to abnormal development of the spore progenitor cell structure wrapped by the cell layer,especially in the double mutant,and the structural integrity of the pollen sac in the mutant anther also appeared abnormal.The results of pollen scanning showed that the pollen formed in the morphologically intact pollen sacs also had abnormal concave outer pollen structures on the surface.The expression of GFP fusion protein was further constructed and found that SSRP1 and SPT16 genes were expressed in the same time and space during flower development,both in the early development of stem apical meristem,calyx primordium,petal primordium and stamen primordium in Arabidopsis thaliana During the process of expression;strong expression in the early stage of anther development,but weak expression in the later stage;protein localization results showed that SSRP1 and SPT16 localized in the nucleus.Real-time quantitative PCR analysis showed that in ssrp1-2 and spt16-2 mutants,the genes related to anther development were up-regulated and down-regulated to different extents,while the genes related to anther development in the ssrp1-2/spt16-2 double mutant were all up-regulated and down-regulated.Severely down-regulated,indicating that FACT plays an irreplaceable and important role in anther development.The expression levels of AG,BAM1 and other genes did not change significantly in ssrp1-2 and spt16-2,but were severely down-regulated in the double mutant,indicating that SSRP1 and SPT16 have redundant functions during anther development.Our results show that FACT is involved in the regulation of early anther development and subsequent microsporocyte development,and plays a very important role in the development of microsporangia and anther structure.2.The laboratory obtained a mutant plant dyi1 under the background of Ler after EMS mutagenesis and subsequent large-scale screening.The phenotype of dyi1 plants is that the pods are short and shriveled without seeds.The mutants and wild-type pollen were crossed,and the F1 generation was all fertile,and fertile plants appeared in the F2 generation:sterile plants=986:301,which was in line with the Mendelian inheritance law,indicating that dyi1 is a single gene-controlled recessive genetic mutant.Further observation found that there were no pollen grains in the mature anthers of the dyi1 mutant;semi-thin sections showed that the tapetum of the dyi1mutant at stage 10 had vacuolation,and there were developmental defects in the development of microspores,and the microspores also had developmental defects.Cavitation occurs.In stage 12 mutants,the tapetum had incomplete degradation of residues,defective microspore development,and finally abortive decline.Transmission section showed that the tapetum was abnormally broken at the ninth stage,and the inner wall of pollen was abnormally developed.The results of anther DIOC₂staining showed that the accumulation of lipids in the tapetum and pollen lining was reduced in dyi1 mutants.Analysis of RNA-Seq data showed that 1509genes were down-regulated in dyi1,including pollen surface proteins and lipid structural proteins were significantly down-regulated in the mutants.These results suggest that DYI1 plays an important role in anther development and may mediate the normal synthesis or transport of lipids,thereby regulating the anther development of plants.In order to determine the location of the DYI1 gene,we preliminarily obtained that the DYI1 gene was located on chromosome 1 of Arabidopsis by map-based cloning.In order to narrow the range,the clone population was expanded for fine mapping,and the DYI1 gene was located between the molecular markers F22K20(28.9M)and T14N5(29.04M).Combined with BSA sequencing,it was found that AT1G77145 was mutated at bases 826 and 827,resulting in the mutation of leucine at position 166 to threonine.At present,the function of this gene has not been reported,and it may be a new anther development gene.