本文選題：水稻 + 基因啟動子��； 參考：《湖南農(nóng)業(yè)大學》2016年碩士論文

【摘要】：啟動子在基因功能與調(diào)控中具有重要的作用,對啟動子的研究有助于我們對基因調(diào)控復雜性的理解。水稻是世界上重要的糧食作物,也是科學研究中的模式植物之一。為探尋可利用的水稻內(nèi)源性啟動子,在前期研究中,本實驗室通過Affymetrix水稻基因表達芯片分析了超級稻兩優(yōu)培九母本培矮64S (Oryza sativa L.)在逆境及正常條件下,不同組織器官在不同的生長發(fā)育時期全基因組的表達差異,篩選到一個在正常條件及逆境條件(干旱、低溫、高溫)下均有較高表達水平的基因OsSG4 (GenBank登錄號：AK068991.1)。在此基礎(chǔ)上,從水稻日本晴基因組DNA中克隆了該基因上游啟動子區(qū)域Ospz4 (1 625 bp)及四個不同長度的5'端缺失片段(長度分別為1293 bp、1 006 bp、834bp、492bp)并構(gòu)建了GUS報告基因植物表達載體。轉(zhuǎn)化農(nóng)桿菌EHA105后,通過注射本生煙草(Nicotiana benthamiana)進行瞬時表達分析,結(jié)果顯示Ospz4啟動了及四個不同長度的5'端缺失片段均能在煙草葉片中驅(qū)動GUS基因的表達,其中最短片段(492 bp)的表達活性最強。為進一步驗證該啟動子在水稻中的活性,以水稻臺北309為受體材料,通過農(nóng)桿菌浸染法獲得各啟動子片段相應的陽性轉(zhuǎn)化植株；經(jīng)GUS組織化學染色驗證和實時熒光定量PCR檢測,實驗結(jié)果顯示：Ospz4啟動子在陽性水稻植株葉片、根、莖、穎花、胚乳及胚芽鞘中均有活性,活性強度為雙35S啟動子的49%;四個不同長度的5'端缺失片段在陽性水稻植株葉片、根、莖、穎殼及T1代幼苗中均有活性,其中長度為492 bp的片段在陽性水稻植株葉片中的活性強度與Ospz4啟動子相當,為雙35S啟動子的48%；長度1 293 bp、1 006 bp和834 bp片段的活性分別為雙35S啟動子的的30%、28%和23%,均明顯弱于全長片段。對轉(zhuǎn)Ospz4啟動子的T1代陽性植株進行逆境處理(干旱、低溫、高溫)后進行熒光定量PCR檢測,結(jié)果表明逆境條件下Ospz4啟動子均能較穩(wěn)定的維持較高活性,分別為正常條件下的112%、94%、105%。本實驗的初步研究結(jié)果表明,Ospz4啟動子是一個類似組成型表達的水稻內(nèi)源性啟動子,在水稻研究中具有一定的應用潛力,并為該啟動子的進一步改造與應用奠定了基礎(chǔ)。
[Abstract]:Promoter plays an important role in gene function and regulation. The study of promoter helps us to understand the complexity of gene regulation. Rice is an important food crop in the world and one of the model plants in scientific research. In order to search for the available rice endogenous promoters, the Affymetrix gene expression microarray was used to analyze the super rice Liangyou peijiu female parent, Pei'ai 64s, Oryza sativa L. Under stress and normal conditions, different tissues and organs expressed different genomes at different stages of growth and development. One of them was selected under normal and stress conditions (drought, low temperature, low temperature). High expression level gene OsSG4 GenBank accession number: AK068991.1. On this basis, the upstream promoter region of Ospz4 1 625 BP and four 5 'terminal deletion fragments (1293 BP 1 006 BP 1 006 bp1 006 BP 834 bp1 992 BP) were cloned from the rice Japanese sunny genomic DNA and the plant expression vector of GUS reporter gene was constructed. After transformation of Agrobacterium tumefaciens EHA105, transient expression analysis was carried out by injecting Nicotiana benthamiana. The results showed that Ospz4 initiated and four 5'terminal deletion fragments of different lengths could drive the expression of GUS gene in tobacco leaves. The expression activity of the shortest fragment 492 BP) was the highest. In order to further verify the activity of the promoter in rice, rice Taipei 309 was used as the recipient material to obtain the corresponding positive transformation plants by Agrobacterium tumefaciens staining, and the results were confirmed by GUS histochemical staining and real-time fluorescence quantitative PCR detection. The results showed that the 1: Ospz4 promoter was active in the leaves, roots, stems, spikelets, endosperm and coleoptile of the positive rice plants, and the activity intensity was 49% of the double 35s promoters, and four 5'terminal missing fragments of different lengths were found in the leaves and roots of the positive rice plants. The activity of stem, glume and T1 generation seedlings was similar to that of Ospz4 promoter, and the length of 492bp fragment was similar to that of Ospz4 promoter in the leaves of positive rice plants. The activity of 1 293 BP 1 006 BP and 834 BP fragment was 30% and 23% of that of the double 35s promoter, respectively, which was significantly weaker than that of the full-length fragment. The T 1 positive plants of transgenic Ospz4 promoter were tested by fluorescence quantitative PCR after stress treatment (drought, low temperature, high temperature). The results showed that the Ospz4 promoter could maintain higher activity under stress conditions, which were 11212994 and 105 under normal conditions respectively. The preliminary results show that Ospz4 promoter is an endogenous promoter similar to the constitutive expression of rice, which has a certain potential for application in rice research, and lays a foundation for the further transformation and application of the promoter.
【學位授予單位】：湖南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2

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本文編號：1953186