水稻亚细胞定位标记系稳定遗传株系培育及其应用初探
发布时间：2018-11-02 10:16
【摘要】：亚细胞定位,是指某种蛋白质或者表达产物在细胞内的具体存在部位。蛋白质的功能与其所在的亚细胞位置密切相关,而亚细胞定位研究可以为确定某些未知蛋白质的功能提供重要的参考信息。在前期研究中,本课题组已经构建了一套保守性定位于主要细胞器的、且携带有特异性荧光蛋白GFP/RFP融合结构的植物表达载体,并初步获得了转基因水稻材料,其中包含的亚细胞定位类型主要有:细胞膜、细胞核、内质网、质体、线粒体、高尔基体、液泡、过氧化物酶体及细胞骨架。本研究进一步对转基因水稻后代材料进行潮霉素检测、PCR及测序鉴定、原生质体荧光观察、叶鞘组织荧光观察,以及转基因水稻株系加代繁殖等,旨在获得稳定遗传的水稻蛋白亚细胞定位参照系材料,并验证标记系材料的应用性。研究结果如下:1.经过潮酶素抗性筛选、PCR验证、原生质体荧光观察及水稻叶鞘组织荧光观察,验证了绝大多数前期所培育的标记系材料能够稳定表达荧光信号,并且正确定位于预测的细胞器;2.针对转化 pCXSN-RFPAtCCASP、pCXSN-GFPmTn、pCXSN-RFPH2B、pCXSN-RFP等结构的荧光信号弱或未检测到荧光信号的材料,重新构建了以Ubiquitin强启动子替代35S启动子驱动荧光蛋白融合结构表达的载体,并进行了验证;3.以稳定表达核定位绿色荧光标记GFP-H2B的水稻为例子,将一个水稻转录因子WRKY45-RFP融合表达结构转化到GFP-H2B水稻原生质体中,荧光观察结果显示WRKY45-RFP和GFP-H2B共定位于细胞核中,表明本研究所培育的荧光标记系能够应用于水稻蛋白的亚细胞共定位研究;4.同样以GFP-H2B水稻为材料,以表达PWL2-mChery-NLS的稻瘟菌株PBV591侵染GFP-H2B水稻叶鞘组织,荧光观察结果显示,PWL2-mCherry-NLS和GFP-H2B共定位于水稻细胞核中,符合前人关于PWL2-mCherry-NLS在稻瘟菌内表达后被转运到水稻细胞核内的研究结果,同样表明水稻荧光标记系在稻瘟-水稻互作研究具有很好的应用前景。
[Abstract]:Subcellular localization refers to the specific location of a protein or expression product in the cell. The function of protein is closely related to the location of its subcellular, and subcellular localization can provide important reference information for determining the function of some unknown proteins. In previous studies, our team has constructed a set of plant expression vectors that are conservatively located in the main organelles and carrying the fusion structure of specific fluorescent protein (GFP/RFP), and obtained transgenic rice materials. The subcellular localization types include cell membrane, nucleus, endoplasmic reticulum, plastid, mitochondria, Golgi body, vacuole, peroxisome and cytoskeleton. In this study, hygromycin detection, PCR and sequencing, protoplast fluorescence observation, leaf sheathing tissue fluorescence observation and transgenic rice line propagation were carried out. The purpose of this study was to obtain rice protein subcellular location reference materials and to verify the applicability of the marker materials. The results are as follows: 1. After screening for hygrogenin resistance, PCR verification, protoplast fluorescence observation and rice leaf sheath tissue fluorescence observation, it was proved that most of the marker materials could stably express fluorescence signals and correctly locate the predicted organelles. 2. In view of the materials with weak or no fluorescence signal transformed into pCXSN-RFPAtCCASP,pCXSN-GFPmTn,pCXSN-RFPH2B,pCXSN-RFP and other structures, the expression vector of the fusion structure of fluorescent protein driven by the strong promoter of Ubiquitin instead of 35s promoter was reconstructed. And verified; 3. The fusion expression structure of a rice transcription factor WRKY45-RFP was transformed into the protoplast of GFP-H2B rice with a stable expression nucleus and green fluorescent labeled rice (GFP-H2B) as an example, and the fusion expression structure of a rice transcription factor WRKY45-RFP was transformed into the protoplast of GFP-H2B rice. The results of fluorescence observation showed that WRKY45-RFP and GFP-H2B were co-located in the nucleus, which indicated that the fluorescent marker lines developed in this study could be used in the subcellular co-localization of rice protein. 4. GFP-H2B rice was also used as a material to infect the leaf sheath of GFP-H2B rice with PBV591 strain expressing PWL2-mChery-NLS. The results of fluorescence observation showed that PWL2-mCherry-NLS and GFP-H2B were co-located in the nucleus of rice. The results of previous studies on the expression of PWL2-mCherry-NLS in rice blast fungus and translocation into rice nucleus also indicated that rice fluorescent marker lines had a good application prospect in rice blast rice interaction.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
本文编号：2305737
[Abstract]:Subcellular localization refers to the specific location of a protein or expression product in the cell. The function of protein is closely related to the location of its subcellular, and subcellular localization can provide important reference information for determining the function of some unknown proteins. In previous studies, our team has constructed a set of plant expression vectors that are conservatively located in the main organelles and carrying the fusion structure of specific fluorescent protein (GFP/RFP), and obtained transgenic rice materials. The subcellular localization types include cell membrane, nucleus, endoplasmic reticulum, plastid, mitochondria, Golgi body, vacuole, peroxisome and cytoskeleton. In this study, hygromycin detection, PCR and sequencing, protoplast fluorescence observation, leaf sheathing tissue fluorescence observation and transgenic rice line propagation were carried out. The purpose of this study was to obtain rice protein subcellular location reference materials and to verify the applicability of the marker materials. The results are as follows: 1. After screening for hygrogenin resistance, PCR verification, protoplast fluorescence observation and rice leaf sheath tissue fluorescence observation, it was proved that most of the marker materials could stably express fluorescence signals and correctly locate the predicted organelles. 2. In view of the materials with weak or no fluorescence signal transformed into pCXSN-RFPAtCCASP,pCXSN-GFPmTn,pCXSN-RFPH2B,pCXSN-RFP and other structures, the expression vector of the fusion structure of fluorescent protein driven by the strong promoter of Ubiquitin instead of 35s promoter was reconstructed. And verified; 3. The fusion expression structure of a rice transcription factor WRKY45-RFP was transformed into the protoplast of GFP-H2B rice with a stable expression nucleus and green fluorescent labeled rice (GFP-H2B) as an example, and the fusion expression structure of a rice transcription factor WRKY45-RFP was transformed into the protoplast of GFP-H2B rice. The results of fluorescence observation showed that WRKY45-RFP and GFP-H2B were co-located in the nucleus, which indicated that the fluorescent marker lines developed in this study could be used in the subcellular co-localization of rice protein. 4. GFP-H2B rice was also used as a material to infect the leaf sheath of GFP-H2B rice with PBV591 strain expressing PWL2-mChery-NLS. The results of fluorescence observation showed that PWL2-mCherry-NLS and GFP-H2B were co-located in the nucleus of rice. The results of previous studies on the expression of PWL2-mCherry-NLS in rice blast fungus and translocation into rice nucleus also indicated that rice fluorescent marker lines had a good application prospect in rice blast rice interaction.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
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