| In flowering plants,female gametophyte development starts from a hypodermal cell at the tip of the nucellus,archesporium cell and then the cell differentiates into a megasporocyte directly.The megasporocyte then undergoes meiosis to give rise to four haploid megaspores.In Arbidopsis thaliana,three of the four megaspores undergo programmed cell death and only the chalazal-most megaspore becomes functional megaspore.The functional megaspore undergoes three rounds nuclear divisions without cytokinesis to form an eight-nucleate embryo sac.After cellularization,the embryo sac is composed of seven cells with four cell types:three antipodal cells at the chalazal end,a diploid central cell with a huge vacuole,two synergid cells and one egg cell at the micropylar end.The process of female gametophyte development involves a series of important developmental biological processes such as cellurization,cell fate determination,cell functional specification.Especially,differential expressed genes involving in the process of female gamete differentiation and double fertilization are extremely important for understanding the molecular mechanism of key questions,which includes double fertilization,zygote genome activation,paternal transcripts and maternal transcripts contributions to embryogenesis and have been focused for a long time in this field.However,limitted by technology,little is known about the answers of these questions.Therefore,it has always been an important project to dissect the cells in the embryo sac.Obviously,effecient isolation of theses cells are essential to cell specific transcriptome analysis,cell-type specific gene screening,in vitro culture and the molecular mechanism of cell fate determination.Although similar works,such as cell isolation and cell culture,have been made in various plant species,little progress has been made in Arabidopsis thaliana,which is the best model plant in plant biology.Therefore,it is necessary and meaningful to make great effort for efficient isolation of the cells in the embryo sac in Arabidopsis.In our project,we tried to isolate all types of cells in the mature embryo sac,and finally we are able to isolate egg cell,synergid cell,central cell,antipodal cell and zygote.Benefit from mRNA extraction and cDNA amplification kit for small samples,we did RNA-seq of egg cells,antipodal cells and zygote.We combined the published early embryo transcriptome data with our sequenced data,then we analyzed these data with bioinformatic tools,which is the basis for the research about the embyo sac component cells and zygote.The related works are list in the following.1.Inspired by previously works,we isolated egg cell,synergid cells,antipodal cells and zygotes with or without enzyme treatment for the first time.Meanwhile,we explored the method of central cell isolation.We explored the optimal procedure for each cell type.Our procedures are able to isolate small amount of cells with high quality,such as egg cells,synergid cells and zygotes in a limited time.We found that central cells are most difficult to be isolated,although we are able to isolate intact central cell,in most cases,we can only isolate the subprotoplast of central cells which lost partial cytoplasm during isolation process.2.We constructed the procedure of mRNA extraction and cDNA amplification of egg cells,zygote and antipodal cells,and the procedure is reliable and repeatable for downstream RNA-seq analysis.The transcriptome data of these cells are essential and valuable for exploring the related scientific questions.We analyzed the transcriptome data of egg cells,antipodal cells and zygotes with bioinformatic tools.And the following are in detail.Firstly,we found that both egg cell and antipodal cell express a large amount of secreted proteins,many of these proteins are extremely important for their functions in fertilization,such as EC1 peptides.Secondly,egg cells and zygotes express much more genes than antipodal cells.Thirdly,egg cells and zygotes express a series of genes which play roles that related with root hair development.Fourthly,we analyzed the transcription factors and kinases expressed in these cells.Lastly,combined with pubilished data on early embryo,we identified a large amount of valuable genes,which may involve in cell fate determination,function specification and zygote genome activation.4.We put a special focus on the egg cell specific genes,and we selectively chose Egg1-7 to analyze their expression pattern.For analyzing the expression pattern of these genes in the root,stem,leaf and flower,we extracted the mRNA from these materials and did RT-PCR to detect Eggl-7's expression level.Meanwhile,we constructed the fusion protein vectors of Egg 1-7 and transgened them into wild type plants to obtain the fusion protein material,and we found that Egg 1-6 are specificly expressed in the egg cell and Egg7 is specifically expressed in the egg cell and pollen.Furthermore,we take Eggl and Egg2 as the example to analyze their detailed expression pattern,we fused Eggl and Egg2's promoter with EGFP-H2B and transgened them into plants,and we found that both Eggl and Egg2 are only expressed in the egg cell nuclei.Then,we isolated egg cells,2-cell proembryos and 32-cell embryos,by small sample RT-PCR,we found that Eggl and Egg2's transcripts were abundant in the egg cell and almost disappear in the two cell embryo and totally disappear in the 32 cell embryo.These results indicate that Egg1 and Egg2 are indeed egg cell specific genes. |
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