铁皮石斛qPCR内参基因的筛选及开花相关基因的表达分析

发布时间：2018-08-31 17:07

【摘要】：铁皮石斛(Dendrobium offcinale Kimura et Migo)隶属兰科(Orchidaceae)石斛属(Dendrobium Sw.),为中国传统药用植物。因其富含多糖、生物碱、菲类化合物等多种药用成分,拥有生津益胃、润肺止咳、增强免疫力等功效。对关键基因进行功能研究有助于更好地开发利用铁皮石斛,基因的表达分析则是基因功能研究的一个重要方面。目前,基因表达量一般通过荧光定量PCR相对定量的方法进行分析,而稳定的内参基因是qPCR相对定量准确的保障。为了研究铁皮石斛不同组织中、不同生长阶段中及非生物胁迫条件下的功能基因表达,筛选特定试验条件下的最佳内参基因或基因组合是十分必要的。本研究选取了 7个常用的内参基因(Actin、18S rRNA、GAPDH、EF-1α、TUA、TUB、UBQ)作为候选基因,通过qPCR绝对定量方法分别比较了铁皮石斛不同组织中(原球茎、根、茎、叶、花、种子)、不同生长阶段以及非生物胁迫条件下(高温、低温和干旱)各候选内参基因的表达差异。随即对获得的数据采用三个内参基因稳定性评估软件geNorm、NormFinder和BestKeeper进行分析,最终筛选到特定试验条件下最合适的内参基因和内参基因数。在铁皮石斛不同组织中,geNorm与NormFinder软件分析结果较为一致,而Bestkeeper软件分析的结果与以上两种软件的分析结果存在差异。geNorm和NormFinder软件分析得出候选内参基因表达稳定性最低者为18S rRNA,最高者为GAPDH。Bestkeeper软件分析得出候选内参基因表达稳定性最低者为TUA,最高者为18S rRNU。此外,依据geNorm软件的配对差异值Vn/n+1,确定该试验所需内参基因的最佳数目是3。综合分析,推荐铁皮石斛不同组织中最佳内参基因组合为EF-1α+Actin +GAPDH。在铁皮石斛不同生长发育阶段,3个软件的分析结果不尽相同,但3个软件分析得出候选内参基因表达稳定性最低者均为TUB,结合geNorm软件推荐的该试验条件下所需内参基因数目是5,同时考虑试验成本和实际可行性,综合推荐基因组合UBQ+GAPDH。在高温(40℃)和低温(4℃)胁迫条件下,geNorm和Bestkeeper软件的分析结果较一致,与NormFinde软件分析结果有所不同。geNorm和Bestkeeper软件分析均得出候选内参基因表达稳定性最低者为TUA ,最高者为18S rRNA。NormFinder软件分析得出候选内参基因表达稳定性最低者为GAPDH,最高者为Actin。结合geNorm软件推荐的该试验条件下所需内参基因是5,同时考虑试验成本和实际可行性,综合推荐基因组合为TUB+18S rRNA。在干旱胁迫条件下,3个内参基因稳定性评估软件的分析结果较一致。3个软件分析均得出候选内参基因表达稳定性最低者为TUA。结合geNorm软件推荐的所需内参基因数目2,综合推荐基因组合18S rRNA+Actin。分别利用筛选得到的最不稳定内参基因和最佳内参基因组合相对定量异戊二烯代谢途径中的关键基因法昵基焦磷酸合酶基因FPS在不同组织和不同生长发育阶段的表达模式。FPS的表达具有组织特异性的特点,在原球茎和种子中表达最高,叶中最低。FPS的表达在不同生长发育阶段存在差异,均呈现先增加后降低的趋势。同时通过绝对定量方法对FPS在上述的试验样品中表达进行分析。不但验证了筛选到的铁皮石斛特定试验条件下最佳内参基因的准确性,而且也探究了FPS在铁皮石斛不同组织中和不同生长发育阶段中的表达模式。基于铁皮石斛基因组,成功克隆了 7个共5类铁皮石斛开花相关的MADS-box基因(FUL、PI、AP3、AG1、AG2、SEP3、AP1)并对其在铁皮石斛花芽、正常花和畸形花中的基因表达进行检测。试验结果显示6个开花相关基因在三种材料中均表达,但表达模式却不相同。开花相关基因在开花过程中表达量显著增强,除AP1外,在畸形花中表达量均显著降低。
[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.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S567.239

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