【作者】 王新超；

【导师】 杨亚军；

【作者基本信息】 中国农业科学院 ， 茶学， 2011， 博士

【摘要】 茶树作为一种多年生植物,在每个年生长周期中,叶芽会以休眠的形式来提高对外界逆境特别是低温的抵抗能力,安全越冬。在春季环境条件合适时,芽打破休眠,开始新一轮的生长周期。本研究以特早生品种龙井43和晚生品种政和大白茶为研究对象,采用抑制消减杂交技术,构建了2个品种休眠芽和萌动芽的2对正、反向cDNA-SSH文库,筛选出一批与休眠或萌发有关的基因,并克隆了与休眠或萌发相关的3个基因的cDNA全长,研究了3个基因和一批与生长素相关的基因在茶树芽休眠到萌发后不同阶段的表达模式,为揭示茶树越冬芽休眠及其解除的分子本质奠定基础。1.经过差示筛选、测序、拼接,获得了1287条独立基因。生物信息学分析结果发现在萌动芽文库中,以核糖体蛋白、组蛋白、叶绿素a/b结合蛋白等基因的表达丰度较高;在休眠芽文库中,以已经登录的茶树休眠芽EST序列、脱水素、冷诱导蛋白等基因的表达丰度较高。GO功能推测及归类结果发现休眠芽和萌动芽在基因表达谱上存在较大差异。2.克隆了茶树生长素抑制蛋白基因CsARP1,CsARP1编码的蛋白质具有Auxin-repressed家族的保守结构域,为水溶性的非分泌型蛋白质,没有信号肽及剪切位点。CsARP1随着休眠程度的加深,表达量增加,在休眠阶段的表达量高于萌发阶段。3.克隆了茶树细胞周期蛋白基因CsCYC1和细胞周期素依赖激酶基因CsCDK1。CsCYC1编码的蛋白质具有细胞周期蛋白的特征信号序列、保守的细胞周期蛋白盒区域和cyclin的典型三维结构。CsCDK1基因编码的蛋白质具有CDK的特征信号序列、保守结构域和CDK的典型三维结构。基因表达结果表明,CsCYC1和CsCDK1的表达是生长期高于休眠期,在由休眠向萌发转变阶段它们的表达量最高。说明这2个基因在茶树芽休眠解除的过程中发挥着重要的作用。与生长素相关基因的片段在茶树芽休眠不同阶段的表达模式研究结果表明:(1)与生长素响应因子同源的6条EST片段中,有5条的表达量是休眠期高于萌发期,另外1条则相反。(2)与生长素原初反应基因同源的4条EST片段的表达模式存在差异,其中与SAURs同源的EST在休眠阶段上调表达,休眠解除以后下调表达。而与GH3同源的3条EST,1条在萌发阶段有一个表达高峰,而在休眠期和萌发以后,表达量都下降。另外2条EST在深休眠期的表达量最高。(3)生长素抑制蛋白类基因在休眠期的表达量高于萌发以后的阶段。(4)与生长素结合类蛋白同源的茶树CsGLP1表达量在萌发期高于休眠期。(5)与生长素内向运输载体基因同源的EST片段在深休眠期的表达量最高。(6)2条受生长素调控的EST表现出不同的表达模式,生长素诱导表达的EST在休眠期的表达量高于萌发期及随后阶段,而另外1条受生长素抑制的EST片段在萌发期的表达远远高于休眠期。这些结果初步说明生长素促进腋芽的休眠。更多还原

【Abstract】 As a perennial plant, tea plant (Camellia sinensis) growth may be influenced by different environment stress, such as low temperature, short daylight during each growth cycle. In winter, its leaf buds need to enter dormancy and this help them to safely pass low temperature. Following the temperature rise and daylight becomes longer, the dormant buds transform to normal growth. In this study, suppression subtractive hybridization was used to construct two pairs of double-barreled cDNA-SSH libraries of bud dormancy and bud break of the two varieties, the special early sprouting cultivar, Longjing 43 and the late sprouting cultivar, Zhenghe Dabaicha. Some dormancy-related or bud break-related genes were isolated, and 3 genes were cloned through RACE method. Gene expression patterns of the 3 genes and some auxin-related genes during the time of bud dormancy and bud break were analyzed through real-time quantitative PCR. It is very helpful to explore the molecular mechanism of bud dormancy in tea plant.1. After screening, the positive clones were sequenced, 1287 unigenes were obtained, which can be classified to 208 contigs and 1079 singletons. The libraries of dormant buds exhibited higher sequence redundancy compare to the ones of sprouting buds no matter species or quantities. And each library has its specific dominant gene expression. In the sprouting bud libraries, the expression of genes related to ribosomal protein, histone, and chlorophyll a/b-binding showed relative higher abundance. While in the dormant bud libraries, the expression of genes related to dormant-related ESTs of tea plant, dehydrin and cold-induced proteins were richness. Based on TAIR gene ontology (GO) classification system, the differential gene expression profiles of dormant and sprouting buds were established. According to the GO results, we found that there were significant differences on gene expression between dormant and sprouting buds. The ratio of genes which associated with the stress response in dormant buds was higher than sprouting buds. But the ratios of genes which associated with growth, cell proliferation, electron transport or energy metabolism in sprouting buds were higher than dormant buds.2. The full length cDNA sequence of the auxin-repressed protein gene (CsARP1) was cloned from dormant buds by SMART RACE-PCR. Homologous alignment showed that the deduced amino acids of CsARP1 contained auxin-repressed domain characteristic and it is hydrophilic protein without signal peptide and transmembrane domains. The results of real-time quantitative PCR showed that the CsARP1 gene was expressed in a higher level in dormant buds than sprouting buds. It suggests that the expression of CsARP1 gene is correlated to the bud dormancy transition.3. The full length cDNA sequences of the cyclin (CsCYC1) and cyclin-dependent kinase (CsCDK1) were cloned from sprouting buds by SMART RACE-PCR. The pupative cyclin of tea plant contained the characteristic cyclin family signature sequence and conservative cyclin-box dormain. The pupative CDK of tea plant contained the characteristic CDK family signature sequence and conservative dormains. The cyclin and CDK of tea plant are all hydrophilic proteins without signal peptides and transmembrane domains. And they have cyclin and CDK typical three dimensional structures, respectively. The results of real-time quantitative PCR showed that the two genes were expressed in a low lever in dormant stages, and then their expression lever increased quickly before sprouting, and after bud break, their expression lever decreased, but it was higher than dormant buds. The results suggested that CsCYC1 and CsCDK1 were correlated to the bud dormancy release.The expression parterns of some auxin-realted genes, including auxin response factors (ARFs), primary auxin responsive genes, auxin-repressed protein genes, auxin-regulated genes, auxin influx carrier gene and auxin-binding protein (ABP) gene, were studied in different stages of tea plant bud dormancy and its release. The results showed that (1) the expression levels of five in six ARF homologous EST fragments were up-regulated in the deep dormancy and down-regulated after sprouted, the level of the last one showed the opposite expression levels. ARF genes were involved in the complex process of tea bud dormancy and its release. (2) There were significant differences among the expression patterns of 4 ESTs which are homologous with primary auxin responsive genes. EST which is homologous with SAURs was up-regulated in the dormancy stage but down-regulated after dormancy release. On the contrary, one of 3 ESTs which are homologous with GH3 was up-regulated in sprouting stage while down-regulated in dormant stages and after sprouting stages. Another two ESTs expressed the highest expression levels in the deep dormancy. One of them showed down-regulated trends in the later dormancy stage and growth stages while the other one showed a small expression peak which lower than that in the deep dormancy. The results indicated primary auxin responsive genes were also involved in the complex process of tea bud dormancy and its release. (3) Expression levels of ARP genes were higher in dormancy stage than that in the post-germination stage. (4) The CsGLP1 gene which was homogenous with ABPs has a lower expression level in the dormancy stage but maintain rising trend after germination. (5) The EST which is homogenous with auxin influx carrier gene had the highest expression levels in the deep dormant stage but maintain lower levels in other stages. (6) Two auxin-regulated ESTs showed different expression patterns. The expression levels of auxin-induced EST were higher in the dormant stages than that in the sprouting stages, while the levels of the other auxin-repressed EST were higher in the sprouting stage than that in the dormant stages. These results indicated auxin promote the dormancy of tea axillary buds.更多还原