茶树越冬芽休眠的分子机理研究

发布时间：2018-05-06 15:04

本文选题：茶树 + 休眠　；参考：《西北农林科技大学》2015年博士论文

【摘要】：茶是世界上消费量最大的饮料,越来越多的研究发现适当饮茶有助于身体健康。茶树是多年生常绿木本植物,除热带茶区外,茶树在秋季开始形成休眠芽以应对冬季的低温胁迫。冬季休眠是茶树的重要生存策略,并影响着茶叶生产及茶叶的经济价值。目前,对茶树休眠机理的研究还鲜有报道。本研究以不同休眠状态的茶树腋芽转录组分析为中心,以生长素和休眠相关基因在茶树休眠中的作用为切入点,深入揭示了茶树越冬芽休眠的分子机理。主要研究结果如下:1.鉴定和筛选了在休眠诱导及激素处理等条件下表达稳定的内参基因。本研究利用6个试验处理共94个试验样品检测了11个内参基因的表达稳定性,并利用4种不同的分析软件对所选基因的表达稳定性进行了综合排序。结果显示,茶树目前常用的内参基因Cs TUBULIN1、Cs ACTIN1、Cs18S r RNA1和Cs UBQ1在几乎所有的试验处理中都表现较差。而Cs PTB1、Cs EF1、Cs SAND1、Cs CLATHRIN1和Cs UBC1是5个最稳定的内参基因,适合在不同试验处理中作为q RT-PCR分析的稳定内参。2.利用RNA-seq技术对处于不同休眠状态的腋芽进行了转录组分析。在不同休眠状态腋芽转录组对比分析中获得了16,125个差异表达基因,在生物学过程聚类中,这些差异表达基因主要集中在细胞过程、单有机体过程、代谢过程、生物学调控、应激反应等类别;在细胞成分聚类中,主要集中在细胞、细胞器、膜和小分子复合体等类别;在分子功能聚类中,主要集中在结合和催化活性、核酸结合转录因子活性、运输活性和结构分子活性等类别。茶树腋芽休眠转换过程中显著差异表达基因鉴定、基因探针富集分析及基因调控网络关联分析显示,植物激素及休眠相关的MADS-BOXs和PEBP家族基因在茶树越冬芽休眠形成和解除中发挥重要作用。3.研究了生长素响应因子Cs ARF1在茶树越冬芽休眠中的作用。利用RACE技术克隆了一编码820个氨基酸的生长素响应因子Cs ARF1。生物信息学分析显示,Cs ARF1编码蛋白具有ARFs家族保守的N端DNA结合域和C端二聚化结构域,且中间区域富含谷氨酸、丝氨酸和亮氨酸,是一个定位于细胞质的具有激活转录功能的可溶性蛋白。Cs ARF1在茶树越冬芽深休眠和萌动期表达量较高,表明该基因可能与茶树越冬芽的休眠维持及解除密切相关。4.揭示了生长素相关基因表达变化与茶树越冬芽休眠形成及解除的联系。为全面了解生长素在茶树休眠中的作用,我们选取12个具有代表性的生长素相关基因,并检测了他们在茶树不同休眠和生长阶段,以及在休眠诱导和生长素拮抗剂处理条件下的表达变化。结果显示,这些基因在不同条件或处理下都有显著的表达差异。生长素浓度测定结果也表明,茶树顶芽和腋芽中的生长素含量均在休眠形成时期明显降低,而在萌芽阶段迅速升高。以上结果进一步证实生长素信号通路在茶树冬季芽休眠转换过程中发挥重要作用。5.茶树Cs FLOWERING LOCUS T(Cs FT)基因具有促进开花和调控生长的双重功能。Cs FT基因是PEBP家族中的重要成员,茶树Cs FT基因的两种转录本(Cs FTa,Cs FTb)被克隆,表达分析显示该基因与茶树休眠转换密切相关。进一步的功能验证表明,在杨树中过表达Cs FTa基因可以强烈地促进转化植株早开花,而Cs FTb基因过表达植株则抑制短日照诱导引起的生长停止和休眠芽的形成。对蛋白结构进行分析发现,Cs FT中的第100位氨基酸可能是与其他调控因子结合,进而调控植物开花的关键因子。6.茶树Cs MADS-BOX基因参与茶树休眠调控。茶树中两个Cs MADS-BOX基因被克隆,并进行了表达分析和功能验证。结果显示,Cs MADS-BOX1基因在休眠形成阶段下调表达,在休眠解除阶段上调表达。在杨树中过表达该基因抑制短日照诱导下的顶端生长点的生长停滞,但在低温诱导条件下过表达植株快速停止生长。这表明MADS-BOX1基因可能是调控茶树顶端生长点生长且对低温敏感的重要基因。
[Abstract]:Tea is the largest consumption of beverages in the world. More and more studies have found that proper tea drink helps health. Tea trees are perennial evergreen woody plants. Apart from the tropical tea area, tea trees begin to form dormant buds in autumn to cope with the low temperature stress in winter. At present, there are few reports on the mechanism of the dormancy of tea trees. In this study, the study of tea tree axillary buds in different dormant states is the center, the role of auxin and dormancy related genes in the dormancy of tea trees is the breakthrough point, and the molecular mechanism of the dormancy of the tea tree is revealed. The main results are as follows: 1. A stable internal reference gene was determined and screened under the conditions of dormancy induction and hormone treatment. In this study, 94 experimental samples were used to detect the stability of the expression of 11 internal reference genes, and the expression stability of the selected genes was synthetically ordered by 4 different analysis software. The results showed that the tea tree was present at present. The internal reference genes Cs TUBULIN1, Cs ACTIN1, Cs18S R RNA1 and Cs UBQ1 are poor in almost all of the experimental treatments. Cs PTB1. A transcriptional analysis of the axillary buds in the state of sleep. 16125 differentially expressed genes were obtained in the contrastive analysis of the axillary buds in different dormancy states. In the biological process clustering, these differentially expressed genes were mainly concentrated in the cell process, single organism process, metabolic process, biological control, stress response and so on. In the class, mainly concentrated in the categories of cells, organelles, membranes and small molecular complexes; in molecular functional clustering, the main focus is on binding and catalytic activity, nucleic acid binding transcription factor activity, transport activity, and structural molecular activity. The identification of significant differential expression in the process of axillary bud dormancy conversion in tea tree and gene probe enrichment analysis And gene regulation network association analysis showed that plant hormones and dormancy related MADS-BOXs and PEBP family genes play an important role in the formation and release of winter bud dormancy of tea tree.3. study the role of auxin response factor Cs ARF1 in the dormancy of tea tree overwintering buds. The use of RACE technology to clone a 820 amino acid auxin response The bioinformatics analysis of Cs ARF1. showed that the Cs ARF1 encoded protein had the conservative N terminal DNA binding domain and the C terminal dimerization domain of the ARFs family, and the intermediate region was rich in glutamate, serine and leucine, which was a soluble protein.Cs ARF1 with the activation function of the cytoplasm in the deep dormancy and germination of the tea tree overwintering buds. The gene expression was high, indicating that the gene may be closely related to the dormancy maintenance and removal of the winter buds of the tea tree..4. revealed the relationship between the changes of auxin related gene expression and the formation and release of the dormancy of the overwintering bud of the tea tree. In order to fully understand the role of auxin in the dormancy of tea tree, we select 12 representative auxin related genes. The results showed that there were significant differences in the expression of these genes under the conditions of dormancy induction and auxin antagonist treatment. The results also showed that the auxin concentration in the apical buds and axillary buds of the tea tree were all closed. The period of dormancy formation was significantly reduced, but it increased rapidly in the bud stage. The above results further confirmed that the auxin signaling pathway plays an important role in the winter bud dormancy transformation of tea tree..5. tea tree Cs FLOWERING LOCUS T (Cs FT) gene has dual function of.Cs FT gene, which promotes flowering and regulation growth, which is an important member of the PEBP family. Two transcripts of the Cs FT gene (Cs FTa, Cs FTb) of the tea tree were cloned. The expression analysis showed that the gene was closely related to the dormancy transformation of the tea tree. Further functional verification indicated that the overexpression of Cs FTa gene in Yang Shuzhong could strongly promote early flowering of the transformed plants, while the Cs FTb gene overexpressed plants inhibited the growth induced by short day illumination. The formation of stopping and dormant buds was analyzed. The analysis of protein structure showed that the 100th amino acids in Cs FT may be combined with other regulatory factors, and then regulate the key factor of plant flowering,.6. tea tree Cs MADS-BOX gene to participate in the regulation of tea dormancy. Two Cs MADS-BOX genes in the tea tree were cloned, and the expression analysis and functional verification were carried out. The results showed that the expression of Cs MADS-BOX1 gene was down regulated at the stage of dormancy formation, and the expression was up-regulated at the stage of dormancy release. The growth of the top growth point under the inhibition of Short Daylight induced by Yang Shuzhong overexpression was stagnant, but the overexpressed plant stopped growing rapidly under the low temperature induction. This suggested that the MADS-BOX1 gene might be the control of the top of the tea tree. An important gene that grows at the tip and is sensitive to low temperature.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S571.1

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