| Maize is a monoecious plant,in which the male and female inflorescence are produced separatedly by the apical meristem and the axillary bud.The process that male inflorescence（tassel）produce pollen grains undergoes a series of complex developmental phases,including that the spikelet meristem and the floral meristem are sequentially differentiated by the male inflorescence meristem,then the generation of anther primordium from floral meristem.Finally,the pollen is produced after 14 stages of development by anther primordium.Thus,the generation of male gametophyte from inflorescence meristem is controlled by accurate and complex regulation of many kinds of gene.Therefore,the research of floral meristem genes can not only clarify the molecular mechanism of the biological development of maize reproductive organs,but also help to explore the regulatory mechanism of male sterility in maize,and further lay a theoretical foundation for hybrid seed production through using male sterile materials.In this study,the bioinformatics analysis,genetic transformation and expression analysis of ZmFM1 gene showed that ZmFM1 was the key floral meristem gene which affected the development of inflorescence,stamen and ovule.In addition,ns1 mutant that affected male inflorescence and anther development were studied through genetic analysis,phenotypic observations and identification of candidate gene.The results showed that ns1 mutant ultimately affected anther fertility due to the mutation of a male floral meristem gene.So as to further determine the mutantion gene and the breeding utilization of ns1 lay the foundation.The results of the two studies have proved the importance of upstream floral meristem genes that affect the anther development,which may not only affect the fertility,an important agronomic character,but also affect the development of the vegetative growth period,leading to the advance of the flowering period.The main results are as follows:1.Through bioinformatics analysis,we found that the promoter of ZmFM1 gene contains hormone,floral meristem,seed specific regulatory elements and abiotic stress response elements such as low temperature and drought;RNA-seq data showed that ZmFM1 gene was highly expressed in tassel and ear;the genes in the interaction network of ZmFM1 contained many genes that affect the development of leaves and floral meristem.2.The phenotypic observations of the ZmFM1 overexpressed Arabidopsis（ZmFM1-OE）showed that the reproductive growth was earlier,the vegetative growth was blocked and the number of branches were reduced.The inflorescence morphology and floral organ characteristics were abnormal,such as the inflorescence top clustered which resulted in the phenomenon of flowers in flowers.The number of anthers increased and homeomorphic transformation occurred,the ovule biological development defects were serious,resulting in low seed setting rate.The results indicated that ZmFM1 was a floral meristem gene that affected the development of stamen,ovule and other floral organs.3.The results of qPCR showed that the expression of GRF9 gene which regulated vegetative development and abscisic acid related gene ABF3 were twice than WT;the expression of floral organ identity gene AG was twice,while the expression of STK gene which regulated the ovule development was less than half than WT.4.Genetic analysis showed that the segregation rate of fertility in F2 population was 3:1,indicating that the male sterile mutant was controlled by recessive single gene and had stable genetic phenomenon.5.Phenotypic observations showed the plant of ns1 mutant was short,the growth of tassel was compact,the number of floral organs of male spike is abnormal which there are three florets and nine anthers in one male spike.When the mature pollen was stained with 1% I2-KI,it could not be stained normally,so the pollen was sterile.6.It was found that the somatic layers of anther locule were disordered in ns1 mutant.The shape of somatic cells were small and irregular.The anther locules were obviously shrunk.7.A candidate gene C1 was screened by three primer method.When the gene was detected in five ns1 mutant plants,both the DNA sequence of C1 gene and the amplified fragment of insertion site were obtained,indicating that the candidate gene C1 was not the candidate gene which caused the male sterility of ns1 mutant. |
