發(fā)布時(shí)間：2018-08-31 17:07

【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(qiáng)免疫力等功效。對關(guān)鍵基因進(jìn)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(dá)分析則是基因功能研究的一個(gè)重要方面。目前,基因表達(dá)量一般通過熒光定量PCR相對定量的方法進(jìn)行分析,而穩(wěn)定的內(nèi)參基因是qPCR相對定量準(zhǔn)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(dá),篩選特定試驗(yàn)條件下的最佳內(nèi)參基因或基因組合是十分必要的。本研究選取了 7個(gè)常用的內(nèi)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(nèi)參基因的表達(dá)差異。隨即對獲得的數(shù)據(jù)采用三個(gè)內(nèi)參基因穩(wěn)定性評估軟件geNorm、NormFinder和BestKeeper進(jìn)行分析,最終篩選到特定試驗(yàn)條件下最合適的內(nèi)參基因和內(nèi)參基因數(shù)。在鐵皮石斛不同組織中,geNorm與NormFinder軟件分析結(jié)果較為一致,而Bestkeeper軟件分析的結(jié)果與以上兩種軟件的分析結(jié)果存在差異。geNorm和NormFinder軟件分析得出候選內(nèi)參基因表達(dá)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(nèi)參基因表達(dá)穩(wěn)定性最低者為TUA,最高者為18S rRNU。此外,依據(jù)geNorm軟件的配對差異值Vn/n+1,確定該試驗(yàn)所需內(nèi)參基因的最佳數(shù)目是3。綜合分析,推薦鐵皮石斛不同組織中最佳內(nèi)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(gè)軟件的分析結(jié)果不盡相同,但3個(gè)軟件分析得出候選內(nèi)參基因表達(dá)穩(wěn)定性最低者均為TUB,結(jié)合geNorm軟件推薦的該試驗(yàn)條件下所需內(nèi)參基因數(shù)目是5,同時(shí)考慮試驗(yàn)成本和實(shí)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(jié)果較一致,與NormFinde軟件分析結(jié)果有所不同。geNorm和Bestkeeper軟件分析均得出候選內(nèi)參基因表達(dá)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(nèi)參基因表達(dá)穩(wěn)定性最低者為GAPDH,最高者為Actin。結(jié)合geNorm軟件推薦的該試驗(yàn)條件下所需內(nèi)參基因是5,同時(shí)考慮試驗(yàn)成本和實(shí)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(gè)內(nèi)參基因穩(wěn)定性評估軟件的分析結(jié)果較一致。3個(gè)軟件分析均得出候選內(nèi)參基因表達(dá)穩(wěn)定性最低者為TUA。結(jié)合geNorm軟件推薦的所需內(nèi)參基因數(shù)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(nèi)參基因和最佳內(nèi)參基因組合相對定量異戊二烯代謝途徑中的關(guān)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(dá)模式。FPS的表達(dá)具有組織特異性的特點(diǎn),在原球莖和種子中表達(dá)最高,葉中最低。FPS的表達(dá)在不同生長發(fā)育階段存在差異,均呈現(xiàn)先增加后降低的趨勢。同時(shí)通過絕對定量方法對FPS在上述的試驗(yàn)樣品中表達(dá)進(jìn)行分析。不但驗(yàn)證了篩選到的鐵皮石斛特定試驗(yàn)條件下最佳內(nèi)參基因的準(zhǔn)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(dá)模式�；阼F皮石斛基因組,成功克隆了 7個(gè)共5類鐵皮石斛開花相關(guān)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_(dá)進(jìn)行檢測。試驗(yàn)結(jié)果顯示6個(gè)開花相關(guān)基因在三種材料中均表達(dá),但表達(dá)模式卻不相同。開花相關(guān)基因在開花過程中表達(dá)量顯著增強(qiáng),除AP1外,在畸形花中表達(dá)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(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;S567.239

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