【作者】 唐忠辉；

【导师】 郑用琏；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 温敏雄性不育系BS366的育性受环境温度控制。在小孢子母细胞至减数分裂的育性敏感时期,将温敏雄性不育系置于适当低温后,温敏雄性不育系表现出花药不开裂、缺少花粉粒等不育表型。然而,温敏雄性不育系的营养生长和雌蕊的发育都表现正常。这表明温敏雄性不育系的小孢子发生过程,相对与营养生长和雌性生殖器官的发育对低温胁迫更为敏感。本研究的目的是探究低温胁迫是如何影响温敏雄性不育系小孢子的发生过程。首先,我们观察温敏雄性不育系败育的细胞学过程。在低温胁迫下,减数分裂Ⅰ时,由于细胞质的异常分裂导致二分体形成异常。激光共聚焦显微镜和透射电镜的进一步观察表明减数分裂Ⅰ时成膜体结构形成异常,细胞板不能正常组装。随后,利用小麦全基因组基因芯片研究雄蕊组织的基因表达谱,分析可育与不育条件下的差异表达基因。研究发现在低温胁迫下,温敏雄性不育系BS366共有840个差异表达基因,其中以育性正常的普通材料Jing411雄蕊组织基因表达谱为对照,筛选出278个普通低温胁迫响应相关基因。比较低温胁迫和对照生长条件下的雄蕊组织基因表达谱,结果表明,在低温胁迫下参与细胞骨架信号传导相关的基因被诱导表达,以响应低温胁迫；但是,参与细胞骨架组装和动态调节等相关的基因被显著抑制。此外,意识到miRNA具有调控生长发育和响应生物或非生物胁迫的多重功能,探究miRNA在雄蕊发育过程中如何响应低温胁迫,将有助于我们在miRNA的基因表达调控层面揭示温敏雄性不育的分子调控机理。通过small RNA的Solexa测序,我们鉴定出80个已知的miRNA,同时,运用降解组测序技术寻找miRNA在雄蕊组织中真实降解的靶基因。miRNA和靶基因的qPCR验证结果表明,调控靶基因ARF的tasiRNA-ARF和miRNA167家族miRNAs受低温胁迫影响,4个ARF靶基因的表达出现异常。因而,推测异常表达的tasiRNA-ARF和miRNA167可能与BS366的温敏雄性不育相关。更多还原

【Abstract】 The male sterility of wheat thermosensitive genic male sterile (TGMS) line BS366 is strictly controlled by temperature. When a TGMS line BS366 is exposed to an appropriate low temperature, starting at the PMC stage and continuing to the meiosis stage, TGMS lines BS366 exhibit indehiscent anthers and lack pollen grains, while other organs, including pistils, and seedlings develop normally. This suggests that microsporogenesis is much more sensitive to cold stress compared with female reproductive development and vegetative growth.Our objective is to elucidate how cold stress influences the microsporogenesis process of TGMS lines BS366. As a first step towards achieving this aim, the histological features were examined in TGMS-line anthers. During cold stress, abnormal separation of dyads occurred during male meiosis I, owing to failure of male meiotic cytokinesis. The fluorescence labeling and transmission electron microscopy results further revealed that the phragmoplast structure was defectively formed and the cell plate was abnormally assembled during meiosis I under cold stress. To gain further insight into the developmental transition throughout microsporogenesis underlying the histological changes, genomic transcriptional profiling of TMGS-line anthers over a developmental time-series was monitored under cold stress and in controls. A total of 840 annotated probe sets showed significant changes in transcript levels under cold treatment, but 278 of these common cold-responsive genes were eliminated by comparisons with the cold-treated Jing411 controls. Comparing the transcriptomes of cold-stressed lines and the controls, signaling systems that orchestrated, cytoskeleton activity were moderately induced in response to cold-stress in anthers of the TGMS line; however, genes involved in dynamic organization of the cytoskeleton were dramatically repressed. Forthermore, microRNA (miRNA) plays an important role in plant development and response to bio-or abio-stress. Exploring the role of miRNA in the anther developmental context to respond to cold stress will help to understand the mechanism of male sterility. Total 80 miRNAs have been identified by small RNA sequencing with next-generation sequencing technology. A transcriptome-wide experimental method, called "degradome sequencing", was used to directly detect cleaved miRNA targets. The expression profiles of miRNAs and their targets indicated that tasiRNA-ARF and miR167 family were significantly depressed by cold stress, however, the ARF target genes were abnormally induced at meiosis stage in TGMS line. These results suggest the concerning relationship between abnormal regulation of miRNAs under cold stress and male sterility of TGMS line.更多还原