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拟南芥VQ20协助WRKY34和WRKY2参与调控花粉发育的机制研究
Alternative TitleArabidopsis thaliana VQ20 assists WRKY34 and WRKY2
雷日华
Subtype博士
Thesis Advisor余迪求
2017-11
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline植物学
Keyword拟南芥 Wrky34 Wrky2 Vq20 Myb97 Myb101 Myb120 雄配子发生
Abstract在开花植物中,雄性配子体(或花粉)通过产生和传递雄性配子到胚珠完成双受精作用,在植物有性殖中起关键作用。植物雄性配子发育受到严格调控,包括复杂和精确转录调控。已经证明WRKY转录因子家族参与拟南芥中的多个生物学过程的调控。WRKY转录因子家族的一些成员常与含有VQ基序的蛋白(VQ蛋白)相互作用,以控制这些生物学过程;然而,WRKY-VQ复合物在植物雄性配子发生中的分子机制在很大程度上仍然是未知的。
我们实验室之前的研究结果表明,冷处理拟南芥野生型诱导花粉中WRKY34表达;相对于野生型花粉,冷处理后wrky34-1花粉表现出花粉活力的增加,萌发速率升高和花粉管伸长。然而,WRKY34介导的花粉发育过程的确切分子机制尚未完全了解。为了研究WRKY34调控这过程的机制,我们通过酵母文库的筛选,鉴定了其潜在的相互作用的蛋白—VQ20。相互作用研究表明,WRKY34和WKRY2转录因子与VQ20蛋白在细胞核内形成转录复合物。进一步的实验证实,VQ20蛋白序列中的保守的VQ基序负责其相互作用。VQ20蛋白定位在细胞核中,并在花粉中特异性表达。表型分析表明WRKY2WRKY34VQ20对花粉发育很重要。w2-1 vq20-1w34-1 vq20-1双突变体没有表现任何可见的发育缺陷。WRKY2WRKY34VQ20的同时突变导致雄性育性降低:花粉发育、花粉萌发和花粉管生长缺陷。机制研究阐明了,VQ20影响了其相互作用的WRKYs蛋白的转录功能。遗传互补实验支持VQ20与WRKY2和WRKY34的相互作用对于花粉发育是必需的,不与WRKY2和WRKY34相互作用的VQ20的突变形式,没有恢复w2-1 w34-1 vq20- 1三突变体植物的育性。进一步芯片分析表明,WRKY2WRKY34VQ20共同调控与花粉发育,花粉萌发和花粉管伸长相关的多个基因的表达。许多受影响的基因参与细胞壁合成,细胞骨架组织,代谢,转运,信号传递和分泌过程。
芯片比较结果说明WRKY34和三个MYB基因在雄配子发育过程中具有相反的表达模式。WRKY2和WKRY34在雄配子发生过程中抑制了三种MYBs基因(MYB97MYB101MYB120)的表达。在雄配子发生过程中,MYB表达量在w2-1 w34-1 vq20-1三突变体中上调。进一步的实验表明,3个MYBs的6个潜在靶基因的表达在w2-1 w34-1 双突变体和w2-1 w34-1 vq20-1三突变体中均明显上调。酵母单杂交和双荧光素酶报告基因实验证明WRKY2和WRKY34通过结合MYB97启动子的W-boxer。当MYB97过表达时,花药无法开裂,花粉不能萌发,影响雄性育性,表现出与w2-1 w34-1 vq20-1三突变体相似的性状。
总而言之,我们的研究提供了证据,VQ20作为WRKY34和WKRY2的重要互作蛋白通过调控植物雄性配子发生中多个花粉发育相关基因(包括MYBs)的表达来参与拟南芥雄性配子的调控。
 
关键词: 拟南芥,WRKY34WRKY2VQ20MYB97MYB101, MYB120, 雄配子发生
Other AbstractIn flowering plants, the male gametophyte (or pollen grain) plays a key role in plant fertility through the generation and delivery male gametes to the embryo sac for double fertilization. Plant male gametogenesis, which is strictly regulated, involves complex and precise regulation of transcriptional reprogramming. WRKY transcription factors have been demonstrated to control a plethora of processes in Arabidopsis. Several members of this family physically interact with VQ motif-containing proteins (VQ proteins) to mediate a plethora of programs; however, the WRKY–VQ complexes in plant male gametogenesis remains largely unknown.
Our previous results have shown that cold treatment increases WRKY34 expression in the wild-type, while wrky34-1 pollen exhibits increased viability, germination efficiency, and pollen tube growth rates in vivo relative to wild-type pollen under cold treatment conditions. Nevertheless, the exact mechanisms underlying WRKY34-mediated processes are still not fully understood. To investigate the mechanisms of WRKY34-mediated processes, we used a yeast two-hybrid assay and identified a potential interacting partners-VQ protein. Further investigation revealed that WRKY34 and WRKY2 interacted with VQ20, and their interaction increased the transcriptional activity of WRKYs. Further experiments revealed that the conserved VQ motif of VQ20 is responsible for their physical interactions. The VQ20 protein localizes in the nucleus and specifically expresses in pollens. Phenotypic analysis showed that WRKY2, WRKY34 and VQ20 are important for pollen development. w2-1 vq20-1 and w34-1 vq20-1 mutant did not exhibit any visible defective phenotypes. Mutations of WRKY2, WRKY34 and VQ20 simultaneously resulted in male sterility, with defects in pollen development, germination, and tube growth. Mechanism investigation revealed that VQ20 affects the transcriptional functions of its interacting WRKY partners. Complementation experiments supported that the interactions of VQ20 with WRKY2 and WRKY34 are essential for pollen development, as a mutant form of VQ20, which failed to interact with WRKY2 and WRKY34, didn’t rescue the phenotypes of the w2-1 w34-1 vq20-1 triple mutant plants. Further expression analysis indicated that WRKY2, WRKY34 and VQ20 co-regulate multiple genes involved in pollen development, germination, and tube growth. Many of the affected genes are involved in cell wall synthesis, cytoskeleton organization, metabolism, transport, signaling, and secretion processes.
WRKY34 and the three MYBs has the converse expression patterns during male gametogenesis. WRKY2 and WKRY34 repress the expression of three MYBs (MYB97, MYB101, and MYB120) during male gametogenesis. MYB expression was up-regulated in the wrky2-1 wrky34-1 vq20-1 triple mutant during male gametogenesis. Further experiments revealed that the expression of six potential targets of the three MYBs were increased in the wrky2-1 wrky34-1 and more markedly up-regulated in wrky2-1 wrky34-1 vq20-1 triple mutant. Yeast one-hybrid and dual luciferase reporter assays were used to demonstrate that WRKY2 and WRKY34 recognize the promoter of MYB97 by binding to its W-boxes. When MYB97 was overexpressed, the anthers failed to dehisce and the pollen was not able to germinate, impacting male fertility.
Taken together, our study provides evidence that VQ20 acts as an important partner of WRKY2 and WKRY34 through regulating expression of multiple pollen development-related genes (including MYBs) in plant male gametogenesis.
 
Keywords: Arabidopsis, WRKY34WRKY2VQ20MYB97MYB101, MYB120, male gametogenesis
Language中文
Document Type学位论文
Identifierhttp://ir.xtbg.org.cn/handle/353005/10697
Collection西双版纳热带植物园毕业生学位论文
Recommended Citation
GB/T 7714
雷日华. 拟南芥VQ20协助WRKY34和WRKY2参与调控花粉发育的机制研究[D]. 北京. 中国科学院研究生院,2017.
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