【作者】 李波；

【导师】 夏秀英；

【作者基本信息】 大连理工大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 芽休眠是指植物生活史中芽生长暂时停顿的现象。落叶果树芽的自然休眠是为抵御冬季的低温环境。芽的休眠特性对物种的保存、繁衍及农业生产都具有重要的意义。自然条件下,进入休眠的植株需要经过一定时间的低温积累才能解除休眠正常萌发生长。低温不足,芽萌发及树体生长发育会出现障碍或异常,严重影响果树生产。蓝莓是具有较高经济价值的新兴果树树种,但由于对其芽休眠及解除机制认识不足,栽培生产中往往出现引种失败、产量低、品质差、设施栽培难以实现等问题,限制了蓝莓的栽培及推广,严重影响了蓝莓产业的发展。本文以蓝莓为试材,研究了花芽休眠进程中主要生理生化指标的变化,并利用RNA-Seq及MSAP等技术分析了蓝莓花芽休眠及解除过程中基因表达的差异及DNA甲基化的变化,从生理、基因转录及转录后调控等角度探究蓝莓休眠及解除的生理及分子机制,挖掘关键基因,为蓝莓分子育种、休眠调控及设施栽培提供指导。主要结果如下：1.休眠期间,蓝莓花芽生理生化指标呈现规律性的变化：脯氨酸含量呈先降后升的趋势,可溶性糖含量及POD活性持续上升,可溶性蛋白含量及SOD活性持续下降。随着休眠的解除,可溶性糖及可溶性蛋白含量、SOD活性均上升,POD活性下降。2.蓝莓内休眠花芽DNA总甲基化率（38.41%）、全甲基化率（27.09%）及半甲基化率（11.32%）均明显高于解除休眠花芽；相对于内休眠花芽,解除休眠花芽约84.48%的位点发生了甲基化模式变化,其中去甲基化位点数和比例分别为248和40.53%,超甲基化的位点数和比率分别为185和30.23%。3.基于RNA-seq技术构建蓝莓内休眠及解除休眠花芽的转录组文库,对不同休眠阶段花芽基因表达谱进行分析。结果表明,相对于内休眠转录组,解除休眠转录组共有935个基因表达发生变化,其中479个基因上调表达,465个基因下调表达。差异表达基因主要参与光合作用、光合天线蛋白、DNA复制及淀粉和蔗糖代谢等生化代谢途径。4.利用qPCR技术分析了Comp₈5212（淀粉代谢过程相关基因）及Comp₇9163 （MADS-box基因）两条差异基因在蓝莓花芽休眠过程中表达量的变化,结果表明,内休眠前,2个基因表达量较低,进入内休眠后表达量上升,内休眠解除前达到最高,内休眠解除后表达量下降。2个基因可能在蓝莓花芽休眠及解除过程中发挥重要作用。5.利用RACE技术从蓝莓芽中克隆得到休眠相关MADS-box同源基因VcDAM1。 qPCR分析表明,该基因在蓝莓花芽内休眠期间相对表达量上调并维持在较高水平,内休眠解除后表达量迅速下降。该基因可能参与了蓝莓花芽休眠及解除的调控。更多还原

【Abstract】 Bud dormancy is a kind of temporarily halt phenomenon in life cycle of plants. Natural bud dormancy of deciduous fruit trees can resist low temperature environment in winter. Bud dormancy has great significance on conservation, propagation and agricultural production of species. In nature conditions, plants need to cultivate in low temperature for a certain time to break dormancy and germinate. Insufficient chilling will block bud germination and growth or lead to abnormal buds, which do have bad influence on fruit production. These will affect the fruit production seriously. Blueberry is a new fruit tree species with high economic value, but the regulation mechanism of bud dormancy is still unknown, therefore, cases of failure in variety introduction, low output, poor quality and lag in facility cultivation technique occurred frequently in blueberry production. These factors hinder the development of blueberry industry seriously.Blueberry floral buds were used as materials, this paper studied the changes of physiological properties and analyzed the variation of gene expression and DNA methylation level and model during dormancy and dormancy release process. This paper aimed to explore the physiological and molecular mechanism of buds dormancy and dormancy release in blueberry, find key regulation genes, and provide guidance for blueberry molecular breeding and dormancy regulation and facility cultivation. The main results were as follows:1. Physiological properties in floral buds of blueberry during dormancy process were varied regularly:the proline content showed a rising trend after an initial drop, the soluble sugar content and the POD activity continued to raise, the soluble protein content and SOD activity continued to decline during endodormancy. With the beginning of dormancy release, the soluble sugar and protein contents, the SOD activity increased, while the POD activity had a decreation.2. The levels of total DNA methylation （38.41%）, hemi-methylation （11.32%） and fully methylation （27.09%） were down-regulated in dormancy released buds. About 84.35% methylation loci varied during dormancy release when compared with dormancy, among which DNA demethylation loci were 243 with percentage of 40.03%, DNA hypermethylation loci were 185 with percentage of 30.48%.3. Based on RNA-seq technology, the endodormancy transcriptome library and dormancy released transcriptome library were built and the digital gene expression profiling of floral buds in different dormancy status were analyzed and compared. The results show that there were 935 genes identified as significant differentially expressed between dormancy bud and dormancy released bud. Among these genes,470 were up-regulated and 465 down-regulated in endodormancy bud. These genes are mainly involved in the photosynthesis, photosynthesis-antenna proteins, DNA replication, starch and sucrose metabolism metabolic pathway.4. Changes in expression quantities of two genes numbered Comp₈5212 （starch metabolic process gene） and Comp₇9163 （MADS-box gene） which expressed differently between dormant and dormancy released floral buds were calculated by quantitative PCR throughout the process of dormancy in 3 blueberry materials. The results showed that the relative expression value of two genes maintained at a low level before endodormancy and increased when enduring endodormancy, achieved to the peak before dormancy release, then decreased after dormancy release. It implied that these two genes might play an important role in the regulation of dormancy and dormancy release in blueberry bud.5. A dormancy-associated MADS-box gene VcDAMl was cloned from dormant buds of blueberry using RACE technology. The dynamic changes of VcDAM1 gene expression in the process of dormancy in floral buds were analyzed by quantitative PCR. The results showed that the relative transcript level of VcDAM1 increased dramatically during endodormancy period and decreased sharply after dormancy releasing. It indicated that VcDAM1 may have relation to regulating bud dormancy and dormancy releasing in blueberry.更多还原