【摘要】：[目的]選擇穩(wěn)定的內(nèi)參基因是準(zhǔn)確分析實時熒光定量PCR(RT-qPCR)結(jié)果的重要前提,本文旨在篩選荷花花瓣著色過程中穩(wěn)定的內(nèi)參基因,使目標(biāo)基因的定量更加準(zhǔn)確。[方法]以荷花4種不同花色(紅、黃、粉、白)品種、不同花發(fā)育期(蕾期、初花期、盛花期、末花期)的花瓣為試材,利用RT-qPCR技術(shù)檢測8個常用看家基因(ACT、EF1α、GAPDH、FPGS、TUA、LEU、CUL和TRY)的表達(dá)水平,并結(jié)合geNorm、NormFinder和BestKeeper軟件對其表達(dá)穩(wěn)定性進(jìn)行評價。為了進(jìn)一步驗證8個候選基因的穩(wěn)定性,分別以它們作為內(nèi)參基因,檢測目的基因查耳酮合成酶基因(CHS)的表達(dá)。[結(jié)果]geNorm軟件分析表明,不同荷花花色品種及不同花發(fā)育期間,EF1α和ACT表達(dá)穩(wěn)定性最好。NormFinder和BestKeeper軟件顯示在不同花色品種中,EF1a和ACT的表達(dá)均較穩(wěn)定;在不同花發(fā)育期間,NormFinder軟件分析顯示ACT表達(dá)最穩(wěn)定,EF1α穩(wěn)定性也相對較高,而BestKeeper軟件分析顯示EF1α表達(dá)最穩(wěn)定,但ACT穩(wěn)定性較差。同時研究發(fā)現(xiàn),GAPDH和TRY在所有樣品中穩(wěn)定性都較差。不同軟件之間結(jié)果的差異可能是由算法不同造成。擬定EF1α和ACT為最適的內(nèi)參基因,進(jìn)一步利用CHS的表達(dá)分析驗證了EF1α和ACT的穩(wěn)定性。[結(jié)論]在荷花不同花色品種及不同花發(fā)育期間,使用EF1α和ACT 2個表達(dá)最穩(wěn)定的基因組合,即可獲得更為精確的基因表達(dá)結(jié)果。本研究結(jié)果對荷花花瓣著色過程中關(guān)鍵基因表達(dá)的RT-qPCR分析具有重要的實用價值。
[Abstract]:[objective] to select stable internal reference gene is an important prerequisite for accurate analysis of real-time fluorescence quantitative PCR (RT-qPCR) results. The purpose of this paper is to screen stable internal reference gene in the process of petal coloration of lotus flower, so as to make the target gene more accurate. [methods] four different flower colors (red, yellow, powder, white) and petals of different flower development stages (bud, early florescence, full flowering, late flowering) were used as the test material. RT-qPCR technique was used to detect the expression levels of eight common housekeeping genes (ACT,EF1 偽 GAPDHN FPGSGAPDHH) and TRY, and the expression stability was evaluated with geNorm,NormFinder and BestKeeper software. In order to further verify the stability of eight candidate genes, they were used as internal reference genes to detect the expression of the target gene Chalcone Synthase gene (CHS). [results] geNorm software analysis showed that the expression stability of EF1 偽 and ACT in different lotus cultivars and different flower development was the best. Norm Finder and BestKeeper software showed stable expression of EF1 偽 and ACT in different flower varieties. Norm Finder software analysis showed that the stability of ACT expression was also relatively high, while that of BestKeeper software was the most stable, but the stability of ACT was poor. At the same time, the stability of GAPDH and TRY in all samples was found to be poor. The difference between different software may be caused by different algorithms. EF1 偽 and ACT were selected as optimal internal reference genes, and the stability of EF1 偽 and ACT was verified by CHS expression analysis. [conclusion] two stable gene combinations, EF1 偽 and ACT, can be used to obtain more accurate gene expression results in different lotus varieties and different flower development periods. The results of this study have important practical value for RT-qPCR analysis of key gene expression in the coloring process of lotus petals.
【作者單位】： 南京農(nóng)業(yè)大學(xué)園藝學(xué)院;江西省觀賞植物遺傳改良重點實驗室/江西省科學(xué)院生物資源研究所;
【基金】：國家自然科學(xué)基金項目(31400600) 江蘇省自然科學(xué)基金項目(BK20140695) 江西省觀賞植物遺傳改良重點實驗室開放基金(2013-KLB-03)
【分類號】：S682.32

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