水稻亞細(xì)胞定位標(biāo)記系穩(wěn)定遺傳株系培育及其應(yīng)用初探
發(fā)布時間：2018-11-02 10:16
【摘要】：亞細(xì)胞定位,是指某種蛋白質(zhì)或者表達產(chǎn)物在細(xì)胞內(nèi)的具體存在部位。蛋白質(zhì)的功能與其所在的亞細(xì)胞位置密切相關(guān),而亞細(xì)胞定位研究可以為確定某些未知蛋白質(zhì)的功能提供重要的參考信息。在前期研究中,本課題組已經(jīng)構(gòu)建了一套保守性定位于主要細(xì)胞器的、且攜帶有特異性熒光蛋白GFP/RFP融合結(jié)構(gòu)的植物表達載體,并初步獲得了轉(zhuǎn)基因水稻材料,其中包含的亞細(xì)胞定位類型主要有:細(xì)胞膜、細(xì)胞核、內(nèi)質(zhì)網(wǎng)、質(zhì)體、線粒體、高爾基體、液泡、過氧化物酶體及細(xì)胞骨架。本研究進一步對轉(zhuǎn)基因水稻后代材料進行潮霉素檢測、PCR及測序鑒定、原生質(zhì)體熒光觀察、葉鞘組織熒光觀察,以及轉(zhuǎn)基因水稻株系加代繁殖等,旨在獲得穩(wěn)定遺傳的水稻蛋白亞細(xì)胞定位參照系材料,并驗證標(biāo)記系材料的應(yīng)用性。研究結(jié)果如下:1.經(jīng)過潮酶素抗性篩選、PCR驗證、原生質(zhì)體熒光觀察及水稻葉鞘組織熒光觀察,驗證了絕大多數(shù)前期所培育的標(biāo)記系材料能夠穩(wěn)定表達熒光信號,并且正確定位于預(yù)測的細(xì)胞器;2.針對轉(zhuǎn)化 pCXSN-RFPAtCCASP、pCXSN-GFPmTn、pCXSN-RFPH2B、pCXSN-RFP等結(jié)構(gòu)的熒光信號弱或未檢測到熒光信號的材料,重新構(gòu)建了以Ubiquitin強啟動子替代35S啟動子驅(qū)動熒光蛋白融合結(jié)構(gòu)表達的載體,并進行了驗證;3.以穩(wěn)定表達核定位綠色熒光標(biāo)記GFP-H2B的水稻為例子,將一個水稻轉(zhuǎn)錄因子WRKY45-RFP融合表達結(jié)構(gòu)轉(zhuǎn)化到GFP-H2B水稻原生質(zhì)體中,熒光觀察結(jié)果顯示W(wǎng)RKY45-RFP和GFP-H2B共定位于細(xì)胞核中,表明本研究所培育的熒光標(biāo)記系能夠應(yīng)用于水稻蛋白的亞細(xì)胞共定位研究;4.同樣以GFP-H2B水稻為材料,以表達PWL2-mChery-NLS的稻瘟菌株P(guān)BV591侵染GFP-H2B水稻葉鞘組織,熒光觀察結(jié)果顯示,PWL2-mCherry-NLS和GFP-H2B共定位于水稻細(xì)胞核中,符合前人關(guān)于PWL2-mCherry-NLS在稻瘟菌內(nèi)表達后被轉(zhuǎn)運到水稻細(xì)胞核內(nèi)的研究結(jié)果,同樣表明水稻熒光標(biāo)記系在稻瘟-水稻互作研究具有很好的應(yīng)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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S511
本文編號：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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S511
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