拟南芥小分子RNA与ARID1协同促进精子形成的分子机制

In contrast to animals, where meiotic products directly become gametes, the germline in plants is established by mitotic divisions after meiosis is completed. The male germline arises by an asymmetric mitotic division (PMI) of each meiotic product. The resulting vegetative and generative cells of the bicellular pollen grain have distinct fates. The larger vegetative cell is arrested at the G1 phase of the cell cycle, while the smaller generative cell divides mitotically (PMII) to produce the two male gametes or sperm cells. Although many genes have been implicated in sperm cell formation, the molecular mechanism as to how the generative cell initiates the second mitosis and divides into two sperm cells remains unclear.DUO POLLEN1 (DUO1) encodes a male germ cell-specific R2R3 Myb transcription factor that is necessary for twin sperm cell formation, mainly through regulating the expression of many male germline-specific genes to dictate cell division and cell specification of the precursor generative cell, thus the duo1 mutant is infertile due to its nonfunctional gametes. In contrast to the myriad DUO1 targets, how DUO1 transcription is regulated is less understood. Only microRNA159 (miR159) was found to inhibit DUO1 expression. One of our previous reports show the anaphase-promoting complex (APC) negatively regulates DUO1 by promoting miR159 biogenesis. However, miR159 is greatly reduced but not absent at the bicellular stage, but DUO1 is obviously activated from the early bicellular stage, indicating that DUO1 activation is not due only to the decrease of miR159 at the bicellular stage and that other factors are required for DUO1 activation during PMII initiation.During investigating epigenetic regulator in sperm cell formation, we have indentified ARID1, an AT-Rich Interacting Domain-containing transcription factor locates in the generative cell before PMII initiation, and the expression of DUO1 is reduced, finally sperm cell formation is failed in the arid1 mutants. Therefore, we hypothesize that ARID1 plays a critical role during DUO1 activation, and ARID1 is necessary for sperm cell formation. Thus, in this proposed study, we will focus on how ARID1 promotes sperm cell formation; how DUO1 is regulated by ARID1; how ARID1 itself is regulated, and what associated components of ARID1 are. The accomplishment of this study will not only broaden our knowledge on how PMII is initiated and regulated during sperm cell formation on the basis of the molecular mechanism of DUO1 activation, and also provide further clues how small RNAs function in spermatogenesis.

植物精子的形成由小孢子经过两次有丝分裂产生。已知DUO1通过激活下游靶基因决定第二次有丝分裂（PM II）的进程，但其调节机制并不清楚。DUO1是miR159的靶标基因，申请人前期的工作表明miR159和DUO1都在PM II时期表达，暗示存在其它因子拮抗miR159激活DUO1。申请人通过生物信息学分析鉴定了转录因子ARID1，并发现arid1突变体中精子形成受阻，DUO1表达降低。亚细胞定位显示ARID1在生殖细胞中表达。因此我们推测ARID1对DUO1的激活起关键作用，是精子形成的重要组分。本课题将深入研究ARID1促进精子形成的调控机理；阐明ARID1调节DUO1的分子机制；解析ARID1的表达调控机制；鉴定ARID1的结合蛋白。该项目的完成将在阐明DUO1的调节机制的基础上加深人们对第二次有丝分裂起始和调控机制的理解，为进一步诠释小分子RNA在精子发生中的作用机制奠定基础。

和动物减数分裂的产物即为生殖细胞不同的是，植物雄性减数分裂的产物小孢子仍需经历两次有丝分裂才能产生成熟的精细胞。小孢子第一次有丝分裂是不对称的，形成一个大的营养细胞(Vegetative Cell)和小的生殖细胞(Generative Cell)；随后小的生殖细胞继续进行有丝分裂产生两个精细胞。已知miR159的靶标基因之一DUO1是花粉第二次有丝分裂的开关基因，但DUO1的调控机制并不清楚。而且，营养细胞在两次有丝分裂过程中需经历染色质去浓缩的过程，但生殖细胞和精细胞的染色质则高度浓缩，但是这种差异性的染色质状态的调控机制也不清楚。第三，营养细胞中转座子被大量激活并产生小RNA，随后小RNA移动至精细胞沉默精细胞的转座子，这种转座子激活和小RNA移动的分子机制也不清楚。项目主持人在项目的支持下，主要围绕模式植物拟南芥中ARID1如何介导精细胞成熟和参与小RNA介导的转座子调控展开研究。通过遗传学、生物化学、细胞生物学和基因组学的手段，项目执行期间，我们取得主要研究成绩有一下几方面：1）发现ARID1通过与miR159拮抗，直接结合DUO1的启动子促进其在二核期开始表达，从而促进花粉的第二次有丝分裂进程（PLoS Genet 2014; Sci China Life Sci 2015）；2）发现ARID1与MET1在生殖细胞中相互抑制，介导了大孢子母细胞细胞命运的决定（JIPB 2017）；3）发现ARID1可能通过与AGO蛋白互作介导花粉中小RNA的移动，从而参与精细胞中转座子的沉默（under review）。这些工作为深入阐明小RNA介导的表观遗传调控在植物精细胞成熟过程中的作用奠定了扎实的基础。