荷花花瓣着色过程实时荧光定量PCR内参基因的筛选及验证

发布时间：2018-09-18 20:38

【摘要】：[目的]选择稳定的内参基因是准确分析实时荧光定量PCR(RT-qPCR)结果的重要前提,本文旨在筛选荷花花瓣着色过程中稳定的内参基因,使目标基因的定量更加准确。[方法]以荷花4种不同花色(红、黄、粉、白)品种、不同花发育期(蕾期、初花期、盛花期、末花期)的花瓣为试材,利用RT-qPCR技术检测8个常用看家基因(ACT、EF1α、GAPDH、FPGS、TUA、LEU、CUL和TRY)的表达水平,并结合geNorm、NormFinder和BestKeeper软件对其表达稳定性进行评价。为了进一步验证8个候选基因的稳定性,分别以它们作为内参基因,检测目的基因查耳酮合成酶基因(CHS)的表达。[结果]geNorm软件分析表明,不同荷花花色品种及不同花发育期间,EF1α和ACT表达稳定性最好。NormFinder和BestKeeper软件显示在不同花色品种中,EF1a和ACT的表达均较稳定;在不同花发育期间,NormFinder软件分析显示ACT表达最稳定,EF1α稳定性也相对较高,而BestKeeper软件分析显示EF1α表达最稳定,但ACT稳定性较差。同时研究发现,GAPDH和TRY在所有样品中稳定性都较差。不同软件之间结果的差异可能是由算法不同造成。拟定EF1α和ACT为最适的内参基因,进一步利用CHS的表达分析验证了EF1α和ACT的稳定性。[结论]在荷花不同花色品种及不同花发育期间,使用EF1α和ACT 2个表达最稳定的基因组合,即可获得更为精确的基因表达结果。本研究结果对荷花花瓣着色过程中关键基因表达的RT-qPCR分析具有重要的实用价值。
[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 伪 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 伪 and ACT in different lotus cultivars and different flower development was the best. Norm Finder and BestKeeper software showed stable expression of EF1 伪 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 伪 and ACT were selected as optimal internal reference genes, and the stability of EF1 伪 and ACT was verified by CHS expression analysis. [conclusion] two stable gene combinations, EF1 伪 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.
【作者单位】：南京农业大学园艺学院;江西省观赏植物遗传改良重点实验室/江西省科学院生物资源研究所;
【基金】：国家自然科学基金项目(31400600) 江苏省自然科学基金项目(BK20140695) 江西省观赏植物遗传改良重点实验室开放基金(2013-KLB-03)
【分类号】：S682.32

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