【作者】 高超；

【导师】 朱本忠；

【作者基本信息】 中国农业大学 ， 食品生物技术， 2014， 博士

【摘要】 果实发育与成熟是一个多基因参与,多途径协同调控的复杂过程。番茄果实成熟过程存在不同水平的调控：DNA水平调控、转录水平调控、转录后水平调控、翻译水平调控以及蛋白水平调控等。Ripening inhibitor (RIN)属于MADS-box家族转录因子,是转录水平调控果实成熟的关键因子之一。microRNA (miRNA)是一类短的非编码RNA,能够在转录后水平调控基因表达,在植物的许多生理生化过程中起着重要的调控作用。本研究采用高通量测序技术,以野生型番茄(AC)和rin突变体的花及果实为材料,研究了RIN、miRNA.果实成熟相关mRNA三者之间的相互作用关系。主要研究结果如下：1.采用高通量测序技术研究了AC和rin突变体花和不同成熟时期果实中基因的表达谱,鉴定到638个有可能受RIN调控的靶基因(431个为RIN直接调控的靶基因),其中372个基因在rin突变体中上调表达,266个基因下调表达。实验进一步对差异表达基因进行分子功能、生物过程和细胞结构三个层次上的功能划分,结果表明RIN蛋白参与调控了多种生理生化过程和代谢途径,如糖代谢、脂类代谢、色素合成、呼吸过程、光合过程、细胞壁代谢、信号物质传递、逆境胁迫、种子发育等等。2.采用高通量测序技术结合分子生物学实验鉴定了AC和rin突变体花及果实中存在的miRNA,并对其在番茄果实成熟过程中和外源乙烯处理条件下的表达模式进行了分析。实验共鉴定了38个家族保守的miRNA和14个家族新的miRNA,其中大部分miRNA在花中表达明显高于果实中表达,并随着番茄果实成熟表达量逐渐降低。19个保守家族的miRNA及其前体基因在rin突变体花及果实差异表达。此外,外源乙烯处理既可以促进miRNA的表达也可以抑制miRNA的表达。3.大部分miRNAs的靶基因为不同家族的转录因子基因；一些miRNAs的靶基因涉及果实成熟过程中各种糖、酸、维生素及风味物质等生物化合物的合成和代谢过程；非高度保守家族miRNAs如miR6022、miR6024、miR6027的靶基因多为防御蛋白基因,表明miRNAs在抵御外界环境胁迫过程中也具有重要功能。此外,miR168和miR403的靶基因为AGO家族基因,进一步证明miRNA在自身合成过程中的作用。4.克隆了番茄RIN基因和缺失MADS盒的RIN基因(tRIN),分别构建了原核表达载体,并以缺失MADS盒的RIN蛋白为抗原获得到了RIN的多克隆抗体。通过染色质免疫共沉淀(Chromatin Immunoprecipitation,CHIP)和凝胶阻滞实验(electrophoretic mobility shift assay, EMSA)证明了RIN蛋白可以在体内及体外结合到MIR172基因启动子区域的顺式作用元件,对miR172的表达起着重要的调控作用。更多还原

【Abstract】 The development and ripening of fruit is a complex process involving many gene regulatory pathways. Ripening inhibitor (RIN) is a vital transcription factor during fruit ripening in tomato that belongs to the MADS-box family. MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level and play important roles in many physiological and biochemical processes. In this study, deep sequencing approach was employed to explore the interplay among RIN, miRNA and ripening-associated mRNA using the flower and tomato of wild type and rin mutant tomato. Main results were as follows:1. Transcriptome profiling of flower and fruit with differenr developmental stage in AC and rin mutant tomato was studied using high-throughput sequencing approach.638genes that targeted by RIN were identified, of which,372genes were upregulated and266genes were downregulated. The functions of the differently expressed genes were categorized according to molecular function, biological process and cellular structure respectively. The results indicated that RIN was involved in regulating many biological processes and metabolic pathways, including carbohydrate metabolism, lipid metabolism, pigment synthesis, respiration process, the photosynthetic process, cell wall metabolism, signaling, response to stress, seed development.2. miRNAs in flower and fruit of AC and rin mutant tomato were identified using high-throughput sequencing approach, and their expression were profiled during fruit ripening and under ethylene treatment. A total of38conserved families of miRNA and14new families of miRNA were identified. Most families of miRNA had a higher expression in flower than that in fruit, and decreased during fruit ripening.19miRNA families and their corresponding precursor genes were differently expressed between wild-type tomato and rin mutant. Additionally, the expressin of part miRNAs were upregulated under ethylene treatment and the other part miRNAs were downregulated.3. The targets of most families of miRNA were transcription factor, and the targets of several miRNAs were all kind of key enzymes involved in glycometabolism, acidmetabolism and flavor biosynthesis. In addition, the targets of part of the nonconserved miRNAs were genes involved in resistance to stress, suggesting that miRNA may play key roles in process againsting to environmental stress. Furthermore, AGO genes were targeted by miR168and miR403, indicating that miRNAs play an important role in the synthesis process of themselves.4. Full length of RIN gene and part RIN gene that without MADS-box were cloned, and the expression vectors were constructed respectively. Polyclonal antibodies was produced using the protein coding by RIN gene without MADS-box. The results of CHIP and EMSA suggested that RIN could bind to the promoter regions of MIR genes, and play important roles in the regulation of miRNA.更多还原