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[1]潘红 赖呈纯** 张静 黄贤贵 林玉玲 赖钟雄**.不同光质条件下刺葡萄红色愈伤组织的RT-qPCR内参基因筛选*[J].应用与环境生物学报,2019,25(06):1-10.[doi:10.19675/j.cnki.1006-687x.2019.01038]
 PAN Hong,,et al.Selection of reference genes for RT-qPCR in red callus of Vitis davidii (Rom. Caill.) Fo?x under different light qualit ies*[J].Chinese Journal of Applied & Environmental Biology,2019,25(06):1-10.[doi:10.19675/j.cnki.1006-687x.2019.01038]
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不同光质条件下刺葡萄红色愈伤组织的RT-qPCR内参基因筛选*()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年06期
页码:
1-10
栏目:
研究论文
出版日期:
2019-12-30

文章信息/Info

Title:
Selection of reference genes for RT-qPCR in red callus of Vitis davidii (Rom. Caill.) Fo?x under different light qualit ies*
作者:
潘红1;?2;?3 赖呈纯2;?3** 张静1;?2;?3 黄贤贵2;?3 林玉玲1 赖钟雄1**
1福建农林大学园艺植物生物工程研究所 福州 350002
2福建省农业科学院农业工程技术研究所 福州 350003
3福建省农产品(食品)加工重点实验室 福州 350003
Author(s):
PAN Hong1;? 2;? 3;? LAI Chengchun2;? 3**;? ZHANG Jing1;? 2;? 3;? HUANG Xiangui2;? 3;? LIN Yuling & LAI Zhongxiong1**
1 Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2 Institute of Agricultural Engineering and Technology, Fujian Academy of Agricultural Sciences , Fuzhou 350003, China
3 Fujian Key Laboratory of Agricultural Product (Food) Processing, Fuzhou 350003, China
关键词:
刺葡萄;?红色愈伤组织;?光质;?内参基因;?实时荧光定量PCR
Keywords:
Vitis davidii (Rom. Caill.) Fo?x;? red callus;? Light quality;? reference genes ;? Real-time quantitative PCR
DOI:
10.19675/j.cnki.1006-687x.2019.01038
摘要:
不同光质对刺葡萄红色愈伤组织(DLR)花青素的积累以及细胞生长具有重要影响。利用RT-qPCR进行不同光质培养的刺葡萄红色愈伤组织功能基因的表达分析时,应筛选适用于不同光质下RT-qPCR分析的内参基因。以9种光质(黑暗、白光、红光、黄光、蓝光、绿光、紫光、暖白光、暖黄光)下培养获得的刺葡萄红色愈伤组织为材料,对10个葡萄中常用的内参基因进行RT-qPCR分析。结果显示,geNorm、Normfinder、BestKeeper等3个内参分析软件因算法不同,内参稳定性排名具有一定差异。geNorm评估排名前5的内参基因为α-Tubulin = AP-2 > 60SRP > EF1-α > UBQ;Normfinder计算排名前5的内参为60SRP > AP-2 > α-Tubulin > EF1-α > GAPDH;BestKeeper 分析的前5个基因依次为UBQ > α-Tubulin > 60SRP > EF1-α > SAND。α-Tubulin、60SRP在3个软件评估中排名最为稳定靠前,NAD5则为最不稳定候选内参基因。拟定α-Tubulin、60SRP为最适内参,对目的基因UFGT进行验证,验证结果显示α-Tubulin、60SRP表达趋势相似,具有较高的稳定性,其组合可获得较为精准的表达分析结果。该研究为后续光质处理刺葡萄红色愈伤组织的基因表达研究奠定基础。
Abstract:
It is an important influence on the accumulation of anthocyanins and cell growth of red callus of Vitis davidii (Rom. Caill.) Fo?x (DLR) by different light qualities. The expression of functional genes of DLR cell cultures under different light quality can be analyzed quickly and accurately using RT-qPCR. In order to improve experimental data accuracy, it is crucial to screen suitable reference genes for RT-qPCR in this specific experiment. The DLR callus were cultured under nine light qualities, including dark, white light, red light, yellow light, blue light, green light, purple light, warm white light, warm yellow light. Then these cell cultures were taken as the materials. Ten reference genes, which were commonly used in grape, were selected as candidate reference genes. And expression stabilities of ten ref erence genes were analyzed by RT-qPCR under nine light qualities.The expression stability ranking of ten candidate reference genes was evaluated by using three soft wares, such as GeNorm, Normfinder and BestKeeper. These soft wares have different algorithms and lead to variation of reference genes rankings . . The top five reference genes of geNorm evaluation were α-Tubulin = AP-2> 60SRP > EF1-α > UBQ. The top five reference genes of Normfinder calculation were 60SRP > AP-2 > α-Tubulin > EF1-α > GAPDH. The top five reference genes of BestKeeper assessment were UBQ > α-Tubulin > 60SRP > EF1-α > SAND. Considering that the stable rankings of ten reference genes by three soft wares, α-Tubulin and 60SRP were the most stable reference genes for normalizing the RT-qPCR data of genes expression of the DLR cell cultures under different light qualities, and NAD5 was the lest stable reference gene. The reliability of α-Tubulin and 60SRP were validated by using the expression of target gene UFGT. The results showed that the expression profiles of UFGT were similar and had high stability using α-Tubulin or 60SRP to normalize , and α-Tubulin + 60SRP combination can achieve more accurate and reliable results . The present study has provided an important reference for analyzing the expression of critical genes in red callus of Vitis davidii under different light qualities.

备注/Memo

备注/Memo:
收稿日期 Received: 2019-01-23 接受日期 Accepted: 2019-05-08
*福建省自然科学基金项目(2016J01126)、福建省农业科学院科技创新团队项目(STIT2017-1-10)和福建农林大学应用基础研究科技创新专项基金(CXZX2017189)资助
**通讯作者 Corresponding authors (E-mail: lccisland@163.com; laizx01@163.com)
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更新日期/Last Update: 2019-05-09