【作者】 崔利；

【导师】 陈劲枫；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2013， 博士

【摘要】 黄瓜(Cucumis sativus L.)在世界各地广泛种植,市场需求巨大。部分的黄瓜品种具有单性结实特性,能在不受粉的情况下正常结实,对减少人工授粉、提高黄瓜产量与品质具有重要意义。许多设施黄瓜栽培品种缺乏单性结实性状,普遍存在坐果受环境影响大、单性结实能力弱等问题,极大地影响了黄瓜的产量和品质,因此选育黄瓜单性结实品种是目前重要的育种目标之一,研究单性结实机理也成为黄瓜分子育种的热点之一,发掘单性结实基因资源,对改良黄瓜栽培品种有着重要的意义。本文主要以黄瓜单性结实品种‘EC1’和非单性结实品种‘8419s-1’为试材,借助RNA-Seq技术发掘与单性结实果实坐果相关的基因,利用基因瞬时表达方法对部分的黄瓜单性结实候选基因进行快速的初步功能验证。另外,选取RNA-Seq分析中表达差异明显的两个生长素受体基因CsTIR1和CsAFB2进行深入研究,明确其在单性结实果实发育中的时空表达变化,分析外源激素对其表达水平的影响,并对CsTIR1基因进行转番茄(Micro Tom)功能验证。最后,本论文还分析了细胞分裂相关基因(CYCD)在单性结实果实发育早期的表达趋势。以上研究取得了以下主要结果：1.利用RNA-Seq技术发掘黄瓜单性结实调控基因及其部分候选基因的功能分析选择开花后2天的幼果,借助RNA-Seq技术,通过比较分析,共获得1339个与单性结实坐果相关的基因,其中759个基因在单性结实坐果中是上调的,580个基因出现下调。在这些差异表达的基因中,与激素相关的基因表达模式支持植物激素是坐果关键因子的观点。选取了其中的12个差异表达基因在单性结实和非单性结实果实发育的早期进行表达水平分析,结果证明所选的基因在单性结实果实中起到重要的调控作用。利用农杆菌介导的瞬时表达方法对10个与生长素、细胞分裂素和赤霉素信号转导相关的,且在黄瓜单性结实果实中特异表达的基因进行功能分析。结果表明,在外源基因导入黄瓜10天后,黄瓜子房,出现以下几种状态：果实膨大、子房停止发育但不萎蔫、“化瓜”。利用GUS assay进行检测,结果显示,除在萎蔫的子房中GUS染色是阴性以外,其他两种状态的子房GUS染色均是阳性,因此推测在蔫子房中外源基因没有瞬时表达。所选的基因当中,与生长素信号相关的两个基因Csa6M125240.1和Csa3M597350.1在黄瓜单性结实形成中起到了负调控作用；细胞分裂素合成相关基因Csa5M166390.1和赤霉素合成相关基因Csa3M015360.1的正向瞬时表达都可以导致黄瓜单性结实的形成。上述研究表明,所选的10个激素信号基因能够诱导单性结实的形成。2. CsTIR1/AFB基因的表达分析及功能研究选择RNA-Seq中差异明显的两个生长素受体基因进行深入研究。结果表明,黄瓜单性结实相关的CsTIR1和CsAFB2分别在黄瓜的子叶和子房中表达量最高,在黄瓜果实发育早期其表达量随着坐果完成逐渐下降,而在没有成功坐果的子房中其表达量随着子房萎蔫程度的加深逐渐上升。NAA处理后6h,CsTIR1和CsAFB2基因的表达量缓慢上调,在处理后9h开始下调,而且随着NAA浓度从5到50μM的增加,CsTIR1和CsAFB2的表达量基本是一致的。CsTIR1在6-BA处理后3h表达量达到最大值,然后开始下降。与CsTIR1不同,CsAFB2的表达量在6h后才开始下降。另外,在GA3的处理下CsTIR1和CsAFB2的反应也不一致,叶片经GA3处理后6h CsTIR1基因表达量达到最大值,而CsAFB2基因在处理后3h到9h时间范围内的表达量几乎没有变化。ABA和乙烯利处理后,这两个基因的表达量都上调。以上结果揭示CsTIR1和CsAFB2在黄瓜果实发育的过程中起到重要的调节作用,其下调表达可能是成功坐果的关键；CsTIR1和CsAFB2基因对外源激素都有反应,虽然是同一个家族但其反应程度和时间不同；ABA和乙烯后其上调表达说明这两个基因可能参与植物的逆境过程。为了研究黄瓜CsTIR1基因的功能,以番茄的子叶为外质体,对黄瓜的CsTIR1基因进行功能验证。结果表明：转超表达CsTIR1基因的番茄出现单性结实的现象,同时转基因番茄表现出植株矮化特点。研究表明黄瓜CsTIR1基因在单性结实形成过程中起到重要的调控作用。3.黄瓜果实发育调控基因CsCYCDs的克隆与表达分析从RNA-Seq数据分析中,获得关于细胞分裂的D型细胞周期蛋白家族基因(CYCD),为研究单性结实果实发育早期的细胞分裂情况,分析了CYCD家族基因在果实发育早期的表达特征。在果实发育早期,CsCYCDs的表达趋势表明天然单性结实果实与授粉果实细胞分裂主要发生在开花后第4天。另外,大部分CYCD基因在天然单性结实果实中的表达模式与授粉果实的一样,但是不同于诱导的单性结实。以上结果表明CYCD基因家族在天然单性结实果实发育早期起着重要的调控作用,使得单性结实的子房不依赖于授粉便可以进行果实发育所必须的细胞分裂。综上所述,黄瓜单性结实果实发育是一个复杂的分子调控过程。本研究发掘了黄瓜单性结实的候选基因,并对其中的部分基因进行功能验证,明确了一些基因在单性结实果实发育过程中的分子调控机理。这为黄瓜单性结实分子调控机制的后续研究奠定了基础。更多还原

【Abstract】 Cucumber (Cucumis sativus L.) is an important vegetable crops widely cultivated around the world, plays an important role in the production and consumption. Due to the low-temperature, low light and high humidity and other factors in the protected cultivation result in poor pollination and fertilization, resulting in abnormal fruit development, thus affecting the economic efficiency of agriculture. Parthenocarpy is an important trait and one of the main breeding objectives in protected cultivation for cucumber. China has rich parthenocarpic cucumber germplasm resources, but many facilities cultivars of cucumber are lack of the parthenocarpic trait, the fruit development affected by the environment, which greatly affected the production and quality of cucumber. Thus, the study on cucumber parthenocarpy, and exploring genes related with parthenocarpy has an important significance for improving cucumber cultivars. In this paper, the genes related with cucumber parthenocarpy were found by the RNA-Seq technology with the cucumber parthenocarpic variety’EC1’and non-parthenocarpic variety’8419s-1’as test materials. Then gene transient expression method was used for rapid functional verification about the part candidate genes of cucumber parthenocarpy. Based on the differentially expressed genes involved in cucumber parthenocarpic fruit development from the analyses of RNA-Seq, two auxin receptor genes CsTIR1and CsAFB2were chosen for the analyses of expressional level in different cucumber organs, and their response to exogenous hormones were studied. In addition, the CsTIR1were over-expressed in tomato for functional verification. The temporal and spatial expression expressional patterns of CYCD genes were also studied for understanding of cell division in the early stage of cucumber parthenocarpic fruit. The main results are as following:1. Using RNA-Seq technology to explore the molecular mechanism of cucumber parthenocarpic fruit set and the preliminary function of genes related with parthenocarpyIn present study, RNA-Seq technique was used to collect the transcriptome data of cucumber fruits2days after anthesis for screening of parthenocarpy genes. Differentially expressed genes (DEGs) were analyzed in both parthenocarpic and pollinated cucumber fruits contrasting with abortive fruits. Trough comparative analyses, a total of1339genes associated with parthenocarpic fruit development were obtained, including759genes up-regulated and580down-regulated. Expression patterns of phytohormone related DEGS supported that phytohormones are key factors for fruit setting. Also selected12of them involved in fruit development process were analyses in early developmental parthenocarpic fruit, and the results indicated that the selected genes play an important regulatory role in parthenocarpic fruit development.The transient expression of gene in plant tissues is a very good method for preliminary research of gene function. In the present study, we used the method of Agrobacterium-mediated transient expression to analysis cucumber parthenocarpic fruit-specific expression of gene function. The results showed that the exogenous gene introduction after10days, Cucumber ovary appeared the following states:expansion fruit, the stopped development ovaries but not wilt and wilt ovaries. GUS assay results in wilting ovary did not carry exogenous gene of Agrobacterium infection to cucumber ovary tissue, so the exogenous genes were not been transformed in ovary tissue. Studies have shown that the selected10genes to stimulate parthenocarpic fruit and this technology can also be used for the function analysis of cucumber fruit characters genes.2. Expression level and function analyses of auxin receptor genes in cucumber parthenocarpic fruit developmentIn our study, CsTIRl and CsAFB2were isolated from RNA-Seq analysis, it were highly expressed in ovaries and leaves and their transcript levels decreased in parthenocarpic and pollinated fruits, but continuously up-regulated in non-parthenocarpic and unpollinated fruits, indicating that down-regulation of CsTIRl and CsAFB2may be necessary for cucumber fruit set and development. The transcript levels of CsTIRl and CsAFB2were significantly induced in leaves by NAA,6-BA, GA3, ABA, and ethephon. The expression levels were up-regulated by ABA and ethephon. These results suggest that CsTIRl and CsAFB2may be involved in abiotic stress response. Thus, CsTIRl and CsAFB2may be important regulators during cucumber fruit development.Auxin receptor TIR1plays an important role in regulating the growth and development of plants. In order to study the function of the cucumber CsTIR1, it was transformed into tomato by Agrobacterium-mediated transformation. The results showed that the transgenic tomato lines overexpressing CsTIR1exhibit parthenocaarpy and dwarf. This proves that the CsTIR1gene may be a key regulator in parthenocarpic fruit development.3. Genome-wide identification of D-type cyclins gene family from cucumber and analysis of their expression patterns in fruit developmentCell division plays an essential role in early fruit development. Plant D-type cyclins (CYCDs) are important regulators in the cell division. However, the knowledge regarding their involvement in early developmental stage of cucumber fruit remains limited. To address this, the cucumber CYCD family genes and their structural characteristics were identified, and then their expressional patterns in early fruit development were analyzed from the anthesis day (0d) to8days after anthesis (8d). The result showed that the cucumber genome contains13different CYCD genes which have been named according to identity percentages of the corresponding orthologs in Arabidopsis and poplar (Populus trichocarpd). The genomic organization of each subgroup cucumber CYCD showed the structure similar to their orthologs in Arabidopsis and poplar. The expression levels of cucumber CYCD genes were investigated in early stage of fruit development under different treatments, including:natural parthenocarpic, pollination, induced parthenocarpic with CPPU treatment. The highest expression level of most of CYCDs appeared at4DAA in natural parthenocarpic and pollinated fruit. Moreover, the expression patterns of most CYCD genes in natural parthenocarpic fruit were nearly coincident with in pollinated fruit, but different from induced parthenocarpic fruit. Collectively, these results will provide molecular insights into the CYCDs which are contributing to the formation of cucumber parthenocarpic fruit.更多还原