| In angiosperms,the pistil is consisted of the stigma,the style and the ovary.The stigma receives the pollen and guide the growth of the pollen tube,the style is the channel for pollen tube growth,and the ovary embeds the embryo sac which is composed of one egg cell,two synergid cells,one central cell with two nuclei,and three or more antipodal cells.When one pollen tube grows into one of the synergid cells and releases two sperm cells,one sperm cell fuses with the egg cell to form the zygote,while the other fuses with the central cell to form fertilizated central cell,then,these fusions lead to the production of the embryo and endosperm,respectively.Thus,the stigma and the embryo sac are essential for the success of the double fertilization.Through the screening of the tissue-specific genes and the analysis of the mutants with defective pistil,researchers got a deep understanding of the molecular regulation mechanism of the pistil development in Arabidopsis.Similar studies were also carried out in maize,and the researchers found that some female gametophyte-specific genes play an important role during the process of the female gametophyte development and the double fertilization.Rice is also one of the most important crops in the world;however,there are very few studies about the pistil development.Therefore,we investigated the mutant dst(defective stigma)by using transcriptome sequencing,ovary staining and clearing,map-based cloning,qRT-PCR and in situ hybridization,and got some results as follows:1.Through the comparison of the gene expression between the stigma and the ovary with the help of the transcriptome sequencing technology,3531 stigma-preferential genes within the pistil were identified.The result of GO analysis showed that the genes related to the cell membrane and pollination were enriched in the stigma while few extracellular region-related genes were preferentially expressed,suggesting that the rice stigma might perform its functions mainly through the stigma pellicle in the attraction,adhesion and germination of the pollen grains.And the genes related to transport,localization,communication and stimulus response were also enriched in rice stigma,implying that secretion might play essential roles in the stigma,which was also confirmed by the GO analysis of the genes related to secretion.Meanwhile,the GO analysis and KEGG analysis displayed the genes related to secondary metabolism were also preferentially expressed in the stigma.Thus,we concluded that during the process of rice pollination,the membrane-related receptors in the stigma received the stimulus signals of the pollen grains and activated the downstream pathways,resulting in the secretion of the effectors(like the secondary metabolites)and response to the stimulus,leading to the germination of the compatible pollen grains.2.We also identified 703 stigma-specific genes and 1257 ovary-specific genes within rice pistil through the analysis of the differentially expressed genes between the stigma and the ovary.The expression analysis of these genes through the rice life history showed that 7 genes were only expressed in the stigma and 45 genes were specially expressed in the ovary.Meanwhile,there were 385 down-regulated genes compared the ovaries without embryo sac in the mutant dst with the normal ovaries in wild type.By detecting their expression in the stigma and the ovary,we found that 122 of them were only present in the ovary.Further,the expression analysis through the rice life history of these 122 genes was done,the results showed that most genes were preferential expressed in the pistil and 14 genes were only expressed in the pistil,suggesting that these genes might play important roles in the pistil.Subsequently,some representive genes were verified using the qRT-PCR and in situ hybridization,showing that the results are similar to the transcriptome sequencing.Besides,the results of the RNA-Seq showed that some reads were clustered far from the known genes,which suggested that there might be novel genes.Using the Cufflinks and CPC softwares,these reads were analysed.Finally,102 novel protein-coding genes were identified,and 6 of them were verified through the semi-quantitative RT-PCR.The identification of the novel protein-coding genes was a complement to the transcriptome of rice,and provided an effective method for the discovery of the novel genes.3.As described above,we identified a rice mutant dst(defective stigma)in our study.The segregation ratio in the progenies of the heterozygotes dst/DST was 3.04:1 and a similar segregation ratio(3.13:1)was got in the F2 population between rice mutant dst and Zhonghua 11,both were close to 3:1,so we thoght that dst was a recessive gene.Using the PCR technology,we found that the phenotype of mutant dst was not co-segregated with T-DNA insertion,suggesting that the phenotype of the mutant dst was not caused by the insertion of the T-DNA,but by the natural mutation.Therefore,the map-based cloning technology was adopted to clone the gene DST.First,the mapping population(F2 population)through the hybridization between rice mutant dst and Zhonghua 11 was constructed,the crossovarate of the SSR markers showed that the crossovarate of RM530,which was located in chromosome 2,was 15.46%,was far below the theoretical value 50%,implying that the mutant gene was located in chromosome 2.And the recombinants of the marker RM530 were different from these of the marker RM561,thus,we inferred that the gene DST was located between the markers RM530 and RM561.To verify our deduction,several other markers in chromosome 2 were selected for experimental analysis,the results showed that the crossovarates of RM3688 and RM3515 were 2.73%and 4.55%,respectively,and their recombinants were different,suggesting that the gene DST was located between them.Further,the SSR markers between RM3688 and RM3515 were screened and their recombinants were analyzed,the recombinants of the markers RM13517 and RM13565 were different,so it is speculated that the mutant gene was located between them.Afterthat,the genome re-sequencing of the F2 population was performed and the analysis of the ratio distribution among the markers confirmed our result.At last,with the help of the SNP markers,we found the mutant gene was located within about 161Kb range between SNP21 and SNP1.There were about 20 genes in the region and the identification of the mutant gene was still on the way.4.During the growth of the mutant dst,it is found that the mutation of the gene DST leaded to pleiotropic phenotypes.During the vegetative growth,the tiller's number of the mutant dst was less than the wild type,implying that DST might be involved in the development of the lateral primordium.After heading,the numbers of the primary branch,the secondary branch and the spikelet in dst were less than that of wild type.Furthermore,there was nothing at many nodes of the dst panicle,where the branches or spikelets were growing in wild type,therefore,it is speculated that the nodes with nothing might be rudimentary branches or spikelets.And in the mature spikelet of the mutant dst,there was no stigma in the pistil while the anthers were faint yellow and shrunken.Through I2-KI staining,it was found that the pollen abortion rate(5.15%)in the mutant dst was very low,but the number of pollen grains was fewer comapared with the wild type.Meanwhile,the results of the FDA staining also showed relatively consistent pollen viability between wild type and the mutant dst,and fewer pollen grains in the mutant dst,implying that a few pollen grains could develop normally in the mutant dst.Besides,the development process of the pistil in the mutant dst was studied by using the staining and transparent technology,the results showed that the mutant dst was similar to the wild type before the biogenesis of the megesporocyte,but the stigma primordium didn't appear.Using the same method,the development of the female gametophytes was also studied and the defect of embryo sac in the mutant dst was due to the absence of the megasporocyte.Therefore,the mutant dst,without the stigma and embryo sac,was an outstanding material to study the gene regulatory network of the rice stigma and embryo sac.Based on the transcriptome analysis of stigma-preferential genes,the molecular basis of stigma specialization and function was investigated and analyzed at the whole genome level,laying the foundation for the following researches of the stigma.Meanwhile,the identification and analysis of the stigma or the embryo sac preferential/specific genes also provided great convenience for the molecular mechanism of the stigma and embryo sac development.Besides,through the analysis of the phenotype of the mutant dst and the cloning of DST gene,the molecular mechanism during the biogenesis of the sigma or the megasporocyte could be investigated,and the regulation mechanism of DST gene in the development process of flower also could be studied,which was very important to reveal the molecular regulatory network in the development process of the rice inflorescence and pistil,thus,our study lay a solid foundation for that. |
PDF Full Text Request