【摘要】：鐵皮石斛(Dendrobium offcinale Kimura et Migo)隸屬蘭科(Orchidaceae)石斛屬(Dendrobium Sw.),為中國傳統(tǒng)藥用植物。因其富含多糖、生物堿、菲類化合物等多種藥用成分,擁有生津益胃、潤肺止咳、增強免疫力等功效。對關鍵基因進行功能研究有助于更好地開發(fā)利用鐵皮石斛,基因的表達分析則是基因功能研究的一個重要方面。目前,基因表達量一般通過熒光定量PCR相對定量的方法進行分析,而穩(wěn)定的內參基因是qPCR相對定量準確的保障。為了研究鐵皮石斛不同組織中、不同生長階段中及非生物脅迫條件下的功能基因表達,篩選特定試驗條件下的最佳內參基因或基因組合是十分必要的。本研究選取了 7個常用的內參基因(Actin、18S rRNA、GAPDH、EF-1α、TUA、TUB、UBQ)作為候選基因,通過qPCR絕對定量方法分別比較了鐵皮石斛不同組織中(原球莖、根、莖、葉、花、種子)、不同生長階段以及非生物脅迫條件下(高溫、低溫和干旱)各候選內參基因的表達差異。隨即對獲得的數據采用三個內參基因穩(wěn)定性評估軟件geNorm、NormFinder和BestKeeper進行分析,最終篩選到特定試驗條件下最合適的內參基因和內參基因數。在鐵皮石斛不同組織中,geNorm與NormFinder軟件分析結果較為一致,而Bestkeeper軟件分析的結果與以上兩種軟件的分析結果存在差異。geNorm和NormFinder軟件分析得出候選內參基因表達穩(wěn)定性最低者為18S rRNA,最高者為GAPDH。Bestkeeper軟件分析得出候選內參基因表達穩(wěn)定性最低者為TUA,最高者為18S rRNU。此外,依據geNorm軟件的配對差異值Vn/n+1,確定該試驗所需內參基因的最佳數目是3。綜合分析,推薦鐵皮石斛不同組織中最佳內參基因組合為EF-1α+Actin +GAPDH。在鐵皮石斛不同生長發(fā)育階段,3個軟件的分析結果不盡相同,但3個軟件分析得出候選內參基因表達穩(wěn)定性最低者均為TUB,結合geNorm軟件推薦的該試驗條件下所需內參基因數目是5,同時考慮試驗成本和實際可行性,綜合推薦基因組合UBQ+GAPDH。在高溫(40℃)和低溫(4℃)脅迫條件下,geNorm和Bestkeeper軟件的分析結果較一致,與NormFinde軟件分析結果有所不同。geNorm和Bestkeeper軟件分析均得出候選內參基因表達穩(wěn)定性最低者為TUA ,最高者為18S rRNA。NormFinder軟件分析得出候選內參基因表達穩(wěn)定性最低者為GAPDH,最高者為Actin。結合geNorm軟件推薦的該試驗條件下所需內參基因是5,同時考慮試驗成本和實際可行性,綜合推薦基因組合為TUB+18S rRNA。在干旱脅迫條件下,3個內參基因穩(wěn)定性評估軟件的分析結果較一致。3個軟件分析均得出候選內參基因表達穩(wěn)定性最低者為TUA。結合geNorm軟件推薦的所需內參基因數目2,綜合推薦基因組合18S rRNA+Actin。分別利用篩選得到的最不穩(wěn)定內參基因和最佳內參基因組合相對定量異戊二烯代謝途徑中的關鍵基因法昵基焦磷酸合酶基因FPS在不同組織和不同生長發(fā)育階段的表達模式。FPS的表達具有組織特異性的特點,在原球莖和種子中表達最高,葉中最低。FPS的表達在不同生長發(fā)育階段存在差異,均呈現先增加后降低的趨勢。同時通過絕對定量方法對FPS在上述的試驗樣品中表達進行分析。不但驗證了篩選到的鐵皮石斛特定試驗條件下最佳內參基因的準確性,而且也探究了FPS在鐵皮石斛不同組織中和不同生長發(fā)育階段中的表達模式。基于鐵皮石斛基因組,成功克隆了 7個共5類鐵皮石斛開花相關的MADS-box基因(FUL、PI、AP3、AG1、AG2、SEP3、AP1)并對其在鐵皮石斛花芽、正�；ê突位ㄖ械幕虮磉_進行檢測。試驗結果顯示6個開花相關基因在三種材料中均表達,但表達模式卻不相同。開花相關基因在開花過程中表達量顯著增強,除AP1外,在畸形花中表達量均顯著降低。
[Abstract]:Dendrobium Offcinale Kimura et Migo belongs to Orchidaceae Dendrobium Sw. It is a traditional Chinese medicinal plant. Because of its rich polysaccharides, alkaloids, Phenanthrenes and other medicinal ingredients, Dendrobium Offcinale Kimura et Migo has the functions of nourishing stomach, moistening lung and cough, and enhancing immunity. Gene expression analysis is an important aspect of gene function research in Dendrobium candidum. At present, gene expression is generally analyzed by fluorescence quantitative PCR, and stable internal reference gene is the guarantee of relative quantitative accuracy of qPCR. In this study, seven common reference genes (Actin, 18S rRNA, GAPDH, EF-1a, TUA, TUB, UBQ) were selected as candidate genes and compared with different groups of Dendrobium candidum by qPCR absolute quantitative method. The expression of the candidate endogenous genes in the tissue (protocorm, root, stem, leaf, flower, seed), at different growth stages and under abiotic stress (high temperature, low temperature and drought) was analyzed by three endogenous gene stability evaluation software geNorm, Norm Finder and Best Keeper, and the data were finally screened under specific experimental conditions. In different tissues of Dendrobium candidum, the results of geNorm and Norm Finder software analysis were consistent, while the results of Bestkeeper software analysis were different from those of the above two software analysis. GAPDH. Bestkeeper software analysis showed that TUA was the lowest and 18S rRNU was the highest. In addition, according to the pairing difference value Vn/n+1 of geNorm software, the optimum number of internal reference genes needed for the experiment was determined as 3. Comprehensive analysis suggested that the best genome combination of internal reference genes in different tissues of Dendrobium candidum was EF-1a+1. Actin+GAPDH.The results of three softwares were different in different growth and development stages of Dendrobium officinale,but the results of three softwares showed that the candidate with the lowest stability of gene expression was TUB,and the number of genes needed under the experimental conditions recommended by geNorm software was 5.Considering the experimental cost and practical feasibility,the results were recommended comprehensively. Genome combination UBQ + GAPDH. The results of geNorm and Bestkeeper software were consistent under high temperature (40 C) and low temperature (4 C), which were different from those of NormFinde software. Both geNorm and Bestkeeper software showed that TUA had the lowest stability of gene expression and 18S rRNA. NormFinder software had the highest stability of gene expression. GAPDH was the lowest and Actin was the highest among the candidates. Considering the cost and feasibility of the experiment, the recommended gene combination was TUB+18S rRNA. The results were consistent. All three software analysis showed that TUA had the lowest stability in the expression of candidate endogenous reference genes. Combined with the number of required endogenous reference genes recommended by geNorm software 2, the genome combination 18S rRNA + Actin was synthetically recommended. The expression pattern of FPS gene, the key gene method, in different tissues and different stages of growth and development. The expression of FPS has tissue-specific characteristics. The expression of FPS is the highest in protocorm and seed, and the lowest in leaf. The expression of FPS is different in different stages of growth and development, showing a trend of first increase and then decrease. Absolute quantitative method was used to analyze the expression of FPS in the above test samples. The results not only validated the accuracy of the best internal reference gene under specific test conditions, but also explored the expression pattern of FPS in different tissues and different growth stages of Dendrobium candidum. Seven MADS-box genes (FUL, PI, AP3, AG1, AG2, SEP3, AP1) related to flowering of Dendrobium candidum were detected and their expression in flower buds, normal flowers and deformed flowers of Dendrobium candidum was detected. The expression level was significantly enhanced, except for AP1, the expression levels in deformities decreased significantly.
【學位授予單位】：南京師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2;S567.239

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