【作者】 李玲；

【导师】 孙越；

【作者基本信息】 华东师范大学 ， 植物学， 2014， 硕士

【摘要】 URO基因属于植物特有的C2H2基因家族,是可以调控植物体内内源生长素动态平衡的转录调控因子。本实验室前期研究结果表明,在拟南芥中,URO基因过量表达可以使植物体内的自由态生长素明显升高,而氨基结合态生长素含量明显下降,进而出现一系列的生长素过量相关的表型特征,例如,顶端优势减弱、植株矮化以及侧枝数增加等。为了充分的研究URO基因的功能及其保守性,我们利用根癌农杆菌介导的方法将拟南芥URO基因转入烟草,获得URO基因烟草过量表达转化子。结果发现,URO基因烟草转化子的表型与拟南芥uro突变体有许多相似之处,包括顶端优势丧失、植株矮化、叶片加厚且卷曲、花型异常、育性下降,光合效率增强等。本研究在前期对于URO基因烟草转化子的表型初步分析的基础上,继续进行转化,扩大了转基因群体,并进一步观察研究了转化子的表型和生理特性,以及基因表达情况。结果发现,URO基因烟草转化子的开花时间推后,衰老延迟。进一步的研究发现转化子体内叶绿素含量明显增多,光合系统Ⅰ和光合系统Ⅱ的效率都增强、叶片细胞明显增大。通过流式细胞仪检测,发现URO基因烟草转化子叶片细胞的多倍体细胞的比例增高。将能够反应生长素信号的pGH3-GUS构建转化URO基因烟草转化子,转化子体内的生长素信号明显强于空载对照,转化子离体叶片经过一周左右的体外培养有不定根出现。因此URO基因的过量表达同样可以导致烟草转化子体内的生长素浓度增强。以上结果说明URO基因的功能在高等植物中有一定的保守性,可以用来进行与生长素含量相关研究和改造工作。另外,转化子的表型暗示URO基因的功能可能与细胞内复制和细胞分裂的调控有关,URO基因的过量表达又可以保持植物叶片较高的光合效率状态,具有一定的应用前景。在本实验室前期工作的基础上,我还对URO基因的可能下游基因URODOWNSTREAM1(UD1)进行了研究。利用构建启动子加报道基因,原位杂交等方法,确定了UD1基因的表达时空特异性。证明了该基因的表达特征与URO基因有较密切的联系。同时,构建了UDl基因过量表达的转化子,并对从Arabidopsis Biological Resource Center获得的T-DNA插入突变体进行了鉴定和初步分析。所有结果都进一步支持UD1是URO基因的下游基因。更多还原

【Abstract】 The transcription factor URO can regulate endogenous auxin homeostasis in plants, which belongs to C2H2 plant-specific gene family. Our previous studies showed that overexpression of URO genes in Arabidopsis leads to a significantly increase of free IAA, and then a series of auxin-related phenotypes appeared. Such as the reduction of apical dominance and plant height and an increase of the number of lateral branches, etc.In order to fully study the function of URO gene and its conservativity, we transfered Arabidopsis URO gene into tobacco by Agrobacterium tumefaciens mediating, which makes tobacco phenotype significant changes, including the reduction of apical dominance and plant height, thicker and more curled leaves, flower abnormalities, decreased fertility, etc. These phenotypes have many similarities to uro mutant in Arabidopsis.On the basis of preliminary analysed of URO tobacco transformants, we Continued to transformate, expanded the populations of transgenic tobacco. Further observations and studies of the phenotypes, physiological characteristics and genes expression of URO tobacco transformants have been done. Our study showed that URO tobacco transformants flowering and senescence were delayed. Further studies showed that transgenic plants’ chlorophyll content and photosynthetic efficiency have increased. In addition, we found that the volume of leaf cells has significantly increased. By flow cytometry, the results showed that the Polyploid cells of URO transformant have increased. The construction of pGH3-GUS can reflect plants’ auxin signal.We transfered it into URO gene transformants, the results showed that the signal of IAA in transformants was stronger than the empty vector control. We cultured the detached leaves of URO gene transformants on hormone-free medium, which can produce adventitious roots. These results proved that overexpression URO gene in tobacco can increase auxin concentration of the transformants.The results above suggest that the function of URO gene in higher plants was conserved and it can be used for research and renovation work of auxin content. In addition, the phenotypes of URO gene transformants suggested that the function of URO gene may take part in endoreduplication and the regulation of cell division. Overexpression of URO gene can maintain a high state photosynthetic efficiency of leaves, which have some application prospect.On the basis of preliminary work, the researchs of URO DOWNSTREAM 1(UD1) gene which may be the URO gene’s downstream gene have been done. According to add reporter gene to UD1’s promoter and in situ hybridization, we identified temporal and spatial expression of UD1 gene which has some Similarity to URO gene. Meanwhile we analyze the UD1 gene overexpressed transformants and loss of function mutant. These results demonstrated that UD1 which belong to GH3 gene famliy is the downstream gene of URO gene.更多还原