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[1]潘红,赖呈纯,张静,等.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):1407-1413.[doi:10.19675/j.cnki.1006-687x.2019.01038]
 PAN Hong,,et al.Selection of reference genes for RT-qPCR from the red callus of Vitis davidii (Rom. Caill.) Fo?x under different light qualities[J].Chinese Journal of Applied & Environmental Biology,2019,25(06):1407-1413.[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:
1407-1413
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 from the red callus of Vitis davidii (Rom. Caill.) Fo?x under different light qualities
作者:
潘红赖呈纯张静黄贤贵林玉玲赖钟雄
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 gene real-time quantitative PCR
分类号:
S663.1 : Q78
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,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. 本研究结果可为后续光质处理刺葡萄红色愈伤组织的基因表达研究提供理论基础. (图5 表2 参39)
Abstract:
Light quality has an important influence on anthocyanin accumulation and the cell growth of the red callus of Vitis davidii (Rom. Caill.) Fo?x (DLR). The expression of functional genes in DLR cell cultures exposed to different light qualities can be rapidly and accurately analyzed using RT-qPCR. In this specific experiment, screening suitable reference genes for RT-qPCR is crucial for improving the accuracy of the experimental data. The DLR calluses were cultured under nine light qualities, including dark, white light, red light, yellow light, blue light, green light, purple light, warm white light, and warm yellow light. Thereafter, the cell cultures were employed as materials. Ten reference genes, which are commonly found in grape, were selected as candidate reference genes, and their expression stabilities were analyzed by RT-qPCR under the different light qualities. The expression stability ranking of the ten candidate reference genes was evaluated using three software, GeNorm, Normfinder, and BestKeeper. These software have different algorithms which lead to variations in reference gene rankings. The top five reference genes were α-Tubulin = AP-2 > 60SRP > EF1-α > UBQ in geNorm; 60SRP > AP-2 > α-Tubulin > EF1-α > GAPDH in Normfinder; and UBQ > α-Tubulin > 60SRP > EF1-α > SAND in BestKeeper. According to the stability rankings of the ten reference genes by the three software, α-Tubulin and 60SRP were the most stable reference genes while NAD5 was the least stable reference gene for normalizing the RT-qPCR data for the gene expression of the DLR cell cultures under different light qualities. The reliability of α-Tubulin and 60SRP was validated using the expression of the target gene, UFGT. The results showed that the expression profiles of UFGT were similar and had high stability using α-Tubulin or 60SRP for normalization, and the combination of α-Tubulin + 60SRP can achieve more accurate and reliable results. The present study provided an important reference for analyzing the expression of critical genes in red callus of Vitis davidii under different light qualities.

参考文献/References:

1 李婉平, 吕晓彤, 刘敏, 覃民扬, 庄席礼, 房玉林. 江西君子谷野生刺葡萄果实品质特性分析[J]. 北方园艺, 2018 (10): 30-40 [Li WP, Lv XT, Liu M, Tan MY, Zhuang XL, Fang YL. Quality characteristics of Vitis davidii Fo?x Berries in gentleman valley of Jiangxi Province [J]. Nor Hort, 2018 (10): 30-40]
2 González-Neves G, Barreiro L, Gila G, Franco J, Ferrer M, Moutounet M, Carbonneau A. Anthocyanie composition of Tannat grapes from the south region of Uruguay [J]. Anal Chem Acta, 2004, 513 (1): 197-202
3 赖呈纯, 范丽华, 黄贤贵, 谢鸿根. 刺葡萄幼胚愈伤组织诱导及其高产原花青素细胞系筛选[J]. 植物生理学报, 2014, 50 (11): 1683-1691 [Lai CC, Fan LH, Huang XG, Xie HG. Callus induction in brier grape (Vitis davidii Fo?x) from immature embryos and screening of cell lines with high-production of oligomeric proanthocyanidins [J]. Plant Physiol J, 2014, 50 (11): 1683-1691]
4 潘红, 赖呈纯, 黄贤贵, 范丽华, 段长青, 李绍振, 赖钟雄. 不同处理对刺葡萄愈伤组织花青素和原花青素生物合成的影响[J].热带作物学报, 2018, 39 (12): 2404-2409 [Pan H, Lai CC, Huang XG, Fan LH, Duan CQ, Li SZ, Lai ZX. Effects of different treatments on anthocyanins and procyanidins biosynthesis in spine grape callus [J]. Chin J Trop Crops, 2018, 39 (12): 2404-2409]
5 景艳军, 林荣呈. 我国植物光信号转导研究进展概述[J]. 植物学报, 2017, 52 (3): 257-270 [Jing YJ, Lin CR. Summary of research progress on plant light signal transduction in China [J]. Chin Bull Bot, 2017, 52 (3): 257-270]
6 Wan HJ , Zhao ZG , Qian CT , Sui YH, Malik AA, Chen JF. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber [J]. Anal Biochem, 2010, 399 (2): 257-261
7 朱海生, 王彬, 陈敏氡, 李永平, 张前荣, 刘建汀, 李大忠, 吴卫东, 温庆放. 中国南瓜内参基因18S rRNA的克隆及其引物开发[J]. 西北农林科技大学学报(自然科学版), 2017, 45 (3): 118-125 [Zhu HS, Wang B, Chen MD, Li YP, Zhang QR, Liu JT, Li DZ, Wu WD, Wen QF. Cloning of 18S rRNA gene from cucurbita moschata and development of its primers [J]. J Nw A F Univ (Nat Sci Ed), 2017, 45 (3): 118-125]
8 Vandesompele J, de Preter K, Pattyn F, Poppe B, Roy NV, de Paepe A, Speleman F. Accurate normalization of real time quantitative RT PCR data by geometric averaging of multiple internal control genes [J]. Genome Biol, 2002, 3 (7): research0034
9 饶慧云, 邵祖超, 柳海宁, 吴月燕, 刘蓉, 李学孚, 李美芹, 钱萍仙. 抗褐化剂对葡萄愈伤组织继代培养过程中酚类物质、相关酶及其基因表达的影响[J]. 植物生理学报, 2015, 51 (8): 1322-1330 [Rao HY, Shao ZC, Liu HN, Wu YY, Liu R, Li XF, Li MQ, Qian PX. Effect of browning inhibitors on callus subculture of phenolic compounds, enzyme and gene expression of grape [J]. Plant Physiol J, 2015, 51 (8): 1322-1330]
10 姚阳春, 刘永胜, 刘明春. 中华猕猴桃‘红阳’Alase家族基因的克隆及表达分析[J]. 应用与环境生物学报, 2017, 23 (2): 209-214 [Yao YC, Liu YS, Liu MC. Cloning and expression analysis of Alase family genes in kiwifruit (Actinidia chinensis) [J]. Chin J Appl Environ Biol, 2017, 23 (2): 209-214]
11 刘生财, 潘君飞, 王 晓, 赵春丽, 赖钟雄, 张梓浩. MeJA对苋菜悬浮细胞类黄酮和类胡萝卜素累积及其代谢相关基因表达的影响[J]. 应用与环境生物学报, 2019, 25 (4): 1-5 [Liu SC, Pan JF, Wang X, Zhao CL, Lai ZX, Zhang XH. Effects of methyl jasmonate on the contents and relate metabolic genes of flavonoids and carotenoids in suspension cells of Amaranthus tricolor [J]. Chin J Appl Environ Biol, 2019, 25 (4): 1-5]
12 Gutierrez L, Mauriat M, GuéninS, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Wuytswinkel OV. The lack of a systematic validation of reference genes: a serious pit fall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants [J]. Plant Biotechnol J, 2008, 6 (6): 609-618
13 Bustin SA. Absolute quantifcation of mRNA using real-time reverse transcription polymerase chain reaction assays [J]. J Mol Endocrinol, 2000, 25 (2): 169-193
14 Tashiro RM, Philips JG, Winefeld CS. Identifcation of suitable grapevine reference genes for qRT-PCR derived from heterologous species [J]. Mol Genet Genomics , 2016, 291 (1): 483-492
15 Lai CC, Pan H, Huang XG, Fan LH, Duan CQ, Li SZ. Validation of reference genes for gene expression analysis of response to anthocyanin induction in cell cultures of Vitis davidii (Rom. Caill.) Fo?x [J]. In Vitro Cell Dev Biol Plant, 2018, 54 (6): 642-657
16 Andersen CL, Jensen JL, ?rntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets [J]. Cancer Res, 2004, 64 (15): 5245-5250
17 Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations [J]. Biotechnol Lett, 2004, 26 (6): 509-515
18 Reddy PS, Reddy DS, Sharma KK, Bhatnagar-Mathur P, Vadez V. Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR [J]. Plant Gene, 2015, 1: 35-42
19 杨丹, 李清, 王贵禧, 马庆华, 朱利泉. 平欧杂种榛实时荧光定量PCR内参基因的筛选与体系建立[J]. 中国农业科学, 2017, 50 (12): 2399-2410 [Yang D, Li Q, Wang GX, Ma QH, Zhu LQ. Reference genes selection and system establishment for real-time qpcr analysis in ping’ou hybrid hazelnut (C. heterophylla Fisch. × C. avellana L.) [J]. Sci Agric Sin, 2017, 50 (12): 2399-2410]
20 Derveaux S, Vandesompele J, Hellemans J. How to do successful gene expression analysis using real-time PCR [J]. Methods, 2010, 50 (4): 227-230
21 Vanguilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis [J]. BioTechniques, 2008, 44 (5): 619-626
22 查倩, 奚晓军, 蒋爱丽, 田益华, 王世平. 葡萄实时定量PCR中稳定内参基因的筛选[J]. 果树学报, 2016, 33 (3): 268-274 [Cha Q, Xi XJ, Jiang AL, Tian HY, Wang SP. Identification of the appropriate reference gene through using a real-time quantitative PCR in grapes [J]. J Fruit Sci, 2016, 33 (3): 268-274]
23 代红军, 秦晨亮, 徐伟荣. 赤霞珠葡萄发育后期RT-PCR内参基因的筛选和验证[J]. 江苏农业学报, 2016, 32 (3): 668-673 [Dai HJ, Qin CL, Xu WR. Screening and validation of reference genes for real-time fluorescence quantitative PCR during grape berry development of Cabernet sauvignon [J]. Jiangsu J Agric Sci, 2016, 32 (3): 668-673]
24 Upadhyay A, Jogaiah S, Maske SR, Kadoo NY, Gupta VS. Expression of stable reference genes and SPINDLY gene in response to gibberellic acid application at different stages of grapevine development [J]. Biol Plantarum, 2015, 59 (3): 436-444
25 李君英, 张鹤, 韩永峰, 邵建柱, 孙建设. 组培条件下苹果实时定量PCR内参基因的筛选[J]. 果树学报, 2016, 33 (9): 1033-1042 [Li JY, Zhang H, Han YF, Shao JZ, Sun JS. Selection of reference genes for teal-time quantitative PCR in apples (Malus domestica) in vitro [J]. J Fruit Sci, 2016, 33 (9): 1033-1042]
26 张计育, 黄胜男, 王涛, 潘德林, 翟敏, 郭忠仁. 金魁猕猴桃RT-qPCR内参基因的筛选[J]. 上海农业学报, 2018, 34 (1): 84-88 [Zhang JY, Huang SN, Wang T, Pan DL, Zhai M, Guo ZR. Screening of reterence genes for reverse transcription quantitative real-time PCR in Actinidia deliciosa [J]. Acta Agric Shanghai, 2018, 34 (1): 84-88]
27 Vera Hernández FP, Martínez Nú?ez M1, Ruiz Rivas M, Vázquez Portillo RE, Bibbins Martínez MD, Luna Suárez S, Rosas Cárdenas FF. Reference genes for RT-qPCR normalization in different tissues, developmental stages and stress conditions of amaranth [J]. Plant Biol, 2018, 20 (4): 713-721
28 Deng LT, Wu LY, Li JC, Ou Yang KX, Ding MM, Zhang JJ, Li SQ, Lin MF, Chen HB, Hu XS, Chen XY. Screening reliable reference genes for RT-qPCR analysis of gene expression in Moringa oleifera [J]. PLoS One, 2016, 11 (8): e0159458
29 魏秀清, 章希娟, 许玲, 陈长忠, 许家辉. 莲雾实时荧光定量PCR内参基因的筛选和验证[J]. 果树学报, 2018, 35 (4): 402-411 [Wei XQ, Zhang XJ, Xu L, Chen CZ, Xu JH. Screening and validation of reference genes for real-time fluorescence quantitative PCR in wax apple [J]. J Fruit Sci, 2018, 35 (4): 402-411]
30 Wei LB, Miao HG, Zhao RH, Han XH, Zhang TD, Zhang HY. Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR [J]. Planta, 2013, 237 (3): 873-889
31 Wool IG. The structure and function of eukaryotic ribosomes [J]. Annu Rev Biochem, 1979, 48 (1): 719-754
32 Mazumder B, Sampath P, Seshadri V, Maitra RK, DiCorleto PE, Fox PL. Regulated release of L13a from the 60S ribosomal subunit as a mechanism of transcript-specific translational control [J]. Cell, 2003, 115 (2): 187-198
33 Clemens MJ. Targets and mechanisms for the regulation of translation in malignant transformation [J]. Oncogene, 2004, 23 (18): 3180-3188
34 Monteiro F, Sebastiana M, Pais MS, Figueiredo A. Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars [J]. PLoS ONE, 2013. 8 (9): e72998
35 Gamm M, Héloir MC, Kelloniemi J, Poinssot B, Wendehenne D, Adrian M. Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis [J]. Mol Genet Genomics, 2011, 285 (4): 273-285
36 Selim M, Legay S, Berkelmann-L?hnertz B, Langen G, Kogel KH, Evers D. Identification of suitable reference genes for real-time RT-PCR normalization in the grapevine-downy mildew pathosystem [J]. Plant Cell Rep, 2012, 31 (1): 205-216
37 刘蓉, 杨国伟, 吴月燕, 饶慧云, 李学孚, 李美芹, 钱萍仙. 光照强度对葡萄愈伤组织形成过程中相关酶活性和基因表达的影响[J]. 生物工程学报, 2015, 31 (8): 1219-1229 [Liu R, Yang GW, Wu YY, Rao HY, Li XF, Li MQ, Qian PX. Effects of light intensity on associated enzyme activity and gene expression during callus formation of Vitis vinifera [J]. Chin J Biotechnol, 2015, 31 (8): 1219-1229]
38 Davis G, Ananga A, Krastanova S, Sutton S, Ochieng JW, Leong S, Tsolova V. Elevated gene expression in chalcone synthase enzyme suggests an increased production of flavonoids in skin and synchronized red cell cultures of North American native grape berries [J]. DNA Cell Biol, 2012, 31 (6): 939-945
39 王彦杰, 陈叶清, 薛泽云, 周华, 金奇江, 徐迎春. 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[J]. 南京农业大学学报, 2017, 40 (3): 408-415 [Wang YJ, Chen YQ, Xue ZY, Zhou H, Jin QJ, Xu YC. Selection and validation of reference genes for RT-qPCR normalization in lotus (Nelumbo nucifera) during petal coloration [J]. J Nanjing Agric Univ, 2017, 40 (3): 408-415]

更新日期/Last Update: 2019-12-25