【作者】 王玲；

【导师】 孙蒙祥； 赵鹏；

【作者基本信息】 武汉大学 ， 发育生物学， 2022， 硕士

【摘要】 与绝大多数动物和裸子植物不同的是,被子植物的一个显著特征是双受精,即成熟花粉中的两个精子(n),一个与卵细胞(n)结合形成受精卵(2n),并进一步发育成胚胎(2n);另一个与中央细胞(2n)融合并发育形成胚乳(3n)。受精作用是联系配子体与孢子体两个世代之间的桥梁。在世代交替的过程中,伴随着由母本转录物控制向合子基因组控制的转变。尽管对这一转变过程已经进行了广泛的研究,但是父本信息在这一过程中的确切作用还所知甚少。一般情况下,未受精的卵细胞不能启动分裂,只有当卵细胞与精细胞融合后,处于静息状态下的卵细胞才能够分裂并启动胚胎发生。因此,必然是存在着某些关键的父本因子(Paternal Factors,PFs),通过受精作用传递到卵细胞中,进而启动卵细胞的分裂。然而,由于研究技术限制,目前尚不清楚究竟是哪些父本因子对于触发拟南芥卵细胞的分裂至关重要。本文研究基于此前发表的卵细胞、精细胞及受精卵的转录组数据,通过对这些数据的系统性分析,从中筛选得到了45个候选父本基因,这些父本基因均在精细胞中表达,卵细胞中不表达,受精卵中有表达。进一步,我们利用卵细胞特异性启动子p EZ3驱动这些父本基因在卵细胞中异位表达,并以卵细胞GFP荧光为指示信号来评估这些父本基因是否表达成功。通过大规模的筛选,有32个父本基因在卵细胞中成功表达,对这些异位表达株系进行去雄处理,结果显示其中31个株系的卵细胞在去雄后不能分裂,而PF27在卵细胞中表达却能够使卵细胞在不受精的情况下自主分裂,形成由四个细胞纵向排列的结构。同样,利用p DD45和p ECS1驱动PF27表达,也能够使卵细胞分裂。此外,我们发现在卵细胞和合子中表达PF27均会导致胚胎模式建成异常。p PF27::PF27-GFP株系的表达模式分析表明PF27在生殖细胞和精细胞中特异表达,而在营养细胞和雌配子体中不表达。将p PF27::PF27-GFP株系的花粉授到p DD45::RFP标记株系的柱头上,结果显示在6 HAP、8 HAP合子中均够明显观察到GFP荧光,这说明PF27蛋白确实能够通过受精作用被传递到卵细胞中。为了进一步探究PF27在卵细胞分裂中的作用,我们通过CRISPR-Cas9创建了kopf27突变体,同时也通过RNAi技术创建了ripf27突变体,表型分析显示pf27突变体精细胞的发育可能存在一定的缺陷,但是其胚胎表型仍有待进一步研究。为了明确PF27异位表达引起的卵细胞命运的改变,我们分离了p EZ3::PF27-GFP异位表达株系中带有GFP荧光的“卵细胞”用于RNAseq,结果表明在这些分裂的“卵细胞”中,与细胞周期和DNA复制相关的基因上调了,小部分合子激活相关基因的表达水平也显著升高。此外,为了探究受精后激活表达基因在胚胎启动过程中的可能作用,我们利用p EZ3启动子在卵细胞中分别表达了WOX2和WOX9,结果显示WOX2和WOX9的表达均不能启动卵细胞的分裂,但都会引起胚胎模式建成的异常。更多还原

【Abstract】 Unlike most animals and gymnosperms,double fertilization is a striking characteristic for angiosperms.It means that of the two sperms(n)in the mature pollen,one fuses with the egg cell(n)to form the zygote(2n),which will develop into the embryo(2n);and the other fuses with the central cell(2n)to develop into the endosperm(3n).Fertilization acts as a bridge to connect the gametophyte and sporophyte generations.During the alternation between two generations,it is accompanied by the transition from maternal transcripts control to zygotic genome control.Although extensive researches about this transition process have been performed,the exact role of paternal factors in this process remains largely unknown.Normally,unfertilized egg cells cannot promote division,only after the egg cell fused with the sperm,can the quiescent egg cell be able to divide and promote the embryogenesis,so there must be some key Paternal Factors(PFs),which are transmitted to the egg cell through fertilization to promote egg cell division.However,due to the technical limitations,it is still unknown which paternal factors are critical for triggering egg cell division in Arabidopsis.Based on the previously published transcriptome data of egg cells,sperm cells and zygotes,we screened out 45 candidate paternal genes through comprehensive analysis.These paternal genes are all expressed in the sperm cells,but absent in the egg cells,maybe in the fertilized egg cells.Next,driven by the egg cell-specific promoters p EZ3,these paternal genes were ectopically expressed in the egg cells,and GFP fluorescence in the egg cells were used as an indicator to evaluate whether these paternal genes are successfully expressed.Through large-scale screening,we finally identified 32 paternal genes that were successfully expressed in the egg cells.We preformed emasculation on these ectopic expression lines,the results showed that the egg cells in 31 ectopic expression lines could not divide after emasculation.However,the ectopic expression of PF27 in egg cell could trigger egg cell division autonomously without fertilization,and form a structure composed of four cells arranged longitudinally.Similarly,the expression of PF27 driven by p DD45 and p ECS1 could also promote egg cell division.In addition,we found that both the expressions of PF27 in egg cell and zygote could result in abnormal embryo patterning.The expression pattern of p PF27::PF27-GFP lines showed that PF27 was specifically expressed in the generative cells and sperm cells,but not in the vegetative cells and female gametophytes.The pistils of p DD45::RFP marker line were pollinated by p PF27::PF27-GFP pollen grains,the results showed that GFP fluorescence could be obviously observed in 6 HAP and 8 HAP zygotes,which indicated that PF27 could indeed be transmitted into the egg cell through fertilization.To further explore the role of PF27 in egg cell division,we obtained kopf27 mutants by CRISPRCas9 and ripf27 mutant by RNAi technology.The results showed that the sperm development was likely impaired in pf27 mutants,but the embryonic phenotype still need to be further studied.In order to clarify the change of egg cell fate caused by the ectopic expression of PF27,we isolated the “egg cells” with GFP fluorescence in the p EZ3::PF27-GFP ectopic expression lines for RNA-seq.The results showed that in these divided “egg cells”,genes related to the cell cycle and DNA replication were up-regulated,while the expression of a small subset of zygotic de novo expressed genes were also upregulated.Furthermore,in order to explore the role of de novo expressed genes after fertilization in promoting egg cell division.WOX2 and WOX9 were expressed in the egg cells driven by p EZ3 respectively,and the results showed that both the expression of WOX2 and WOX9 could not trigger egg cell division,but caused abnormal embryo patterning.更多还原