【作者】 张文；

【导师】 彭雄波；

【作者基本信息】 武汉大学 ， 发育生物学， 2020， 硕士

【摘要】 高等植物的受精是世代转换的一个转折点,此时会产生两个平行事件:花粉管释放的一个精细胞会与胚囊中的卵细胞融合,未来发育为二倍体的胚胎,另一个精细胞与中央细胞融合,发育成为胚胎发育提供营养的三倍体胚乳。卵细胞受精后发育为合子,随着合子的分裂形成胚,胚进一步发育成植株。卵细胞向合子发育的转换是早期胚胎发生过程中遗传控制的关键点,它代表了合子的发育不再依赖于卵细胞提供的母本因子,而是由合子控制。合子基因激活对应于基因组水平上合子从头转录的开始,它是卵细胞向合子发育的转换发生的条件之一。合子激活的基因对于胚胎发育具有非常重要的作用但由于技术的限制,合子激活基因的分子机制并没有得到进一步深入的研究。本论文在拟南芥转录组数据的基础上,筛选出了卵细胞不表达,而合子表达的转录因子ZYGOTE1(ZYG1)和ZYG2,以这两个基因的拟南芥突变体植株作为研究材料,通过表达模式、突变体表型分析、恢复实验及启动子甲基化水平检测等实验,探究ZYG1和ZYG2基因在拟南芥早期胚胎发育过程中的作用机理。取得的主要结果如下:1.合子激活基因的鉴定和筛选:通过本实验室前期取得的卵细胞、合子的转录组测序结果,从中筛选出了11个基因,这些基因在卵细胞内没有表达,但在合子中高表达。通过转基因对这些基因在胚胎发育过程的表达情况进行了进一步的分析,筛选出了合子从头转录的转录因子ZYG1和其同源基因ZYG2。2.ZYG1和ZYG2融合蛋白在受精后胚胎表达:通过观察proZYG1::ZYG1-GFP和proZYG2::ZYG2-GFP在胚胎和成熟组织内的表达模式,发现GFP信号在胚胎受精前没有表达,受精后在合子、二胞、四胞至球形期、心形期、鱼雷期及子叶前期均有表达,子叶后期主要集中表达在根尖。3.ZYG1和ZYG2参与早期胚胎发育:筛选出ZYG1和ZYG2的突变体,观察到任一基因单突变不能引起角果败育。通过杂交获得zyg1-1 zyg2-1/+,zyg1-2/+zyg2-2两种突变体,发现其受精后角果内存在败育的胚珠,且败育胚珠与正常胚珠比例大约为1:3,符合孟德尔遗传分离定律。在这两种突变体子代中不能分离到纯合双突变体,表明双突变导致胚胎致死。对zyg1-1 zyg2-1/+,zyg1-2/+zyg2-2中的败育胚珠进行透明观察,发现胚胎的早期发育受阻。4.ZYG1和ZYG2不是印记基因:为验证ZYG1和ZYG2是否具有印记效应,将两个基因的融合蛋白模式转基因植株与野生型正反交,观察授粉后1至2天胚胎内的荧光信号的表达,发现转基因植株无论作为母本还是父本均不影响荧光信号在胚胎内的表达。5.ZYG1启动子没有明显的甲基化修饰:为验证ZYG1的特异表达模式是否由于其启动子活性受到甲基化控制,我们分离了具有ZYG1表达活性的根尖组织和不表达ZYG1的叶片,采用亚硫酸氢盐法对两个样品中的ZYG1启动子区域进行了甲基化分析。结果表明ZYG1启动子没有明显的甲基化修饰。6.ZYG1的下游信号初探:构建proZYG1::LEC1-GFP,转基因至zyg1-2/+zyg2-2突变体植株中,通过观察胚胎内的荧光信号的表达,筛选到有LEC1表达的突变体植株。对其是否能够恢复突变体表型还需要进一步的观察。更多还原

【Abstract】 Fertilization of higher plants is a turning point in the generational transition.During fertilization two parallel events occur: sperm and egg cells fuse to form a diploid embryo,and another sperm fuses with the two polar nuclei of the central cell to form a triploid endosperm.The egg cell develops into a zygote after fertilization.The zygote develops into an embryo that further develops into a mature plant.The transition from egg cells to zygote development is a key point of genetic control during early embryogenesis.It represents that the development of zygote no longer depends on maternal factors provided by egg cell,but is controlled by zygote.Genome-wide gene activation in the zygote,termed zygotic gene activation,is one of the conditions for transition from egg cells to zygote.However,the gene activated by zygote plays a very important role in embryo development.However,due to technical limitations,the molecular mechanism of these genes has not been further studied.Based on the results obtained in previous work about transcriptome of egg cells and zygotes,the zygote-activated transcription factors ZYG1 and ZYG2 were identified.The Arabidopsis plants with mutant on these two genes were used as research object to investigate the functions of ZYG1 and ZYG2 genes in the early development process of embryos.By studying the expression pattern of ZYG1 gene,phenotype of mutant plants after hybridization,recovery experiments,and promoter methylation verification,etc.,an attempt was made to explore the role of ZYG1 gene in the early embryonic development of Arabidopsis thaliana.The experimental results were summarized as follows:1.Identification and screening of zygote-activated genes: 11 genes were selected through our laboratory’s transcriptome sequencing results of egg cells and zygotes obtained in the previous period.These genes were not expressed in egg cells,but were highly expressed in zygotes.The expression of these genes during embryonic development was further analyzed by transgene,and the de novo zygote transcription factor ZYG1 and its homologous gene ZYG2 were screened.2.ZYG1 and ZYG2 fusion proteins expressed in embryo after fertilization: By observing the expression patterns of proZYG1::ZYG1-GFP and proZYG2::ZYG2-GFP in embryos and mature tissues,it was found that the GFP signal was not expressed before fertilization,and expressed all phases after fertilization,like zygote,two-celled embryo,four-celled embryo,globular-stage embryo,heart-stage embryo,torpedo-stage embryo,early cotyledon,and mainly expressed in the root tip at the later stage of cotyledon.3.ZYG1 and ZYG2 are involved in early embryo development: mutants on ZYG1 and ZYG2 were screened out,and it was observed that single mutant could not cause seeds abortion.zyg1-1 zyg2-1/+ and zyg1-2/+ zyg2-2 were obtained through the cross in a reciprocal manner.It was found that the numbers of seeds abortion obeys law of segregation and affects early embryo development4.ZYG1 and ZYG2 are not imprinted genes: In order to verify whether ZYG1 and ZYG2 have the imprinting effect,the transgenic plants of the fusion protein model vector of the two genes are crossed with wild-type plants,and the embryos within 1 to2 days after pollination are observed.The expression of the fluorescent signal,it was found that the transgenic plants did not affect the expression of the fluorescent signal in the embryo,either as a maternal or a paternal parent.5.ZYG1 promoter has no obvious methylation modification :To verify whether the specific expression pattern of ZYG1 is controlled by methylation due to its promoter activity,we isolated the root tip tissue that express ZYG1 and the leaves that did not express ZYG1.The bisulfite method was used to analyze the methylation of the ZYG1 promoter region of two samples.The results showed that the ZYG1 promoter had no obvious methylation modification.6.Preliminary exploration of downstream signaling of ZYG1: construct p ZYG1:LEC1-GFP,transgene it into zyg1-2/+ zyg2-2 mutant plants,and observe the expression of fluorescence signals in the embryo.Observing the expression of fluorescence signals in embryos,we selected mutant plants with LEC1 expression.更多还原