茶叶科学 >

2016 , Vol. 36 >Issue 1: 92 - 101

DOI: https://doi.org/10.13305/j.cnki.jts.2016.01.012

不同氮处理茶树实时定量PCR内参基因筛选和验证

  • 刘圆 ,
  • 王丽鸳 ,
  • 韦康 ,
  • 成浩 ,
  • 张芬 ,
  • 吴立赟 ,
  • 胡娟
展开
  • 中国农业科学院茶叶研究所 国家茶树改良中心,浙江 杭州 310008
刘圆,男,硕士研究生,主要从事茶树分子生物学研究。

收稿日期: 2015-04-14

  网络出版日期: 2019-08-23

基金资助

浙江省茶树农业新品种选育重大科技专项（2012C12905）、国家茶叶产业技术体系（nycytx-23）

收起

Screening and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Tea Plant (Camellia sinensis) under Different Nitrogen Nutrition

  • LIU Yuan ,
  • WANG Liyuan ,
  • WEI Kang ,
  • CHENG Hao ,
  • ZHANG Fen ,
  • WU Liyun ,
  • HU Juan
Expand
  • Tea Research Institute of the Chinese Academy of Agricultural Sciences, National Center for Tea Improvement, Hangzhou 310008, China

Received date: 2015-04-14

  Online published: 2019-08-23

Fold

摘要

为筛选不同氮处理下茶树（Camellia sinensis）实时荧光定量PCR（qRT-PCR）试验体系中最佳内参基因,以茶树幼叶、成熟叶和幼根为材料,应用qRT-PCR分析GAPDH、18S rRNA、β-actin、RPL13、α-tubulin、RuBP等6个内参基因在不同氮浓度和氮源处理下的表达变化,借助GeNorm和NormFinder程序对候选内参基因稳定性进行评价。结果表明,在不同氮浓度和氮源处理下,GAPDH、β-actin和RPL13表达稳定性较好,GAPDH+β-actin组合稳定性最佳,可用作茶树基因表达研究的内参基因;而α-tubulin和RuBP表达稳定性较差,不适合做内参基因。为进一步证实上述结果,分别以GAPDH、α-tubulin 、GAPDH+β-actin组合为内参基因,分析茶树幼叶中硝酸根转运蛋白基因（CsNRT1.2）和铵根转运蛋白基因（CsAMT1.1）的表达水平,发现以GAPDH和GAPDH+β-actin组合为内参基因时,目的基因的相对表达量随处理时间延长变化规律基本一致;而以α-tubulin为内参基因时,目的基因的变化规律与前两者存在明显差别。因此,根据特定试验体系选择合适的内参基因对于qRT-PCR定量结果的准确性和可靠性具有重要意义。

关键词： qRT-PCR; 茶树; 氮处理; 内参基因

本文引用格式

刘圆 , 王丽鸳 , 韦康 , 成浩 , 张芬 , 吴立赟 , 胡娟 . 不同氮处理茶树实时定量PCR内参基因筛选和验证[J]. 茶叶科学, 2016 , 36(1) : 92 -101 . DOI: 10.13305/j.cnki.jts.2016.01.012

Abstract

The objective of this study was to select the most reliable reference genes for qRT-PCR analysis of target tea plant genes under varying nitrogen source and availability. We chose 6 housekeeping genes which included five commonly used and one new candidates to systematically assess their expression levels at three different tissues (young leaves, mature leaves and roots) under different nitrogen regimes by qRT-PCR. GeNorm and NormFinder software were used to analyze and evaluate the data for reference genes. The results indicated that GAPDH, β-actin and RPL13 are the best reference genes for normalizing target gene expression in tea plant under different nitrogen nutrition, whereas α-tubulin and RuBP are not suitable in many experimental conditions and the best combination (GAPDH+β-actin) was recommended. Meanwhile, the expression levels of CsNRT1.2 and CsAMT1.1 in young leaves of tea plants were analyzed. The results showed that the variation tendency of CsNRT1.2 and CsAMT1.1 are exactly consistent when using GAPDH and GAPDH+β-actin as reference genes. However, the expression levels of these genes are showed significant differences when α-tubulin was used as a reference gene. Thus, validation of suitable reference genes for specific condition can guarantee the accurate quantification of the target genes in qRT-PCR analysis.

Key words： nitrogen treatment; reference gene; qRT-PCR; tea plant (Camellia sinensis)

参考文献

[1] Bustin S A.Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): Trends and problems[J]. Journal of Molecular Endocrinology, 2002, 29(1): 23-39.
[2] Bustin S A.Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. Journal of Molecular Endocrinology, 2000, 25(2): 169-193.
[3] Radonić A, Thulke S, Mackay I M, et al.Guideline to reference gene selection for quantitative real-time PCR[J]. Biochemical and Biophysical Research Communications, 2004, 313(4): 856-862.
[4] Gutierrez L, Mauriat M, Guenin S, et al.The lack of a systematic validation of reference genes: A serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants[J]. Plant Biotechnology Journal, 2008, 6(6): 609-618.
[5] Brunner A M, Yakovlev I A, Strauss S H.Validating internal controls for quantitative plant gene expression studies[J]. BMC Plant Biol, 2004, 4:14.
[6] Gutierrez L, Mauriat M, Pelloux J, et al.Towards a systematic validation of references in real-time RT-PCR[J]. Plant Cell, 2008, 20(7): 1734-1735.
[7] Thellin O, Zorzi W, Lakaye B, et al.Housekeeping genes as internal standards: Use and limits[J]. Journal of Biotechnology, 1999, 75(2/3): 291-295.
[8] Warzybok A, Migocka M.Reliable reference genes for normalization of gene expression in cucumber grown under different nitrogen nutrition[J]. PLoS One, 2013, 8(9): e72887.
[9] Manoli A, Sturaro A, Trevisan S, et al.Evaluation of candidate reference genes for qPCR in maize[J]. Journal of Plant Physiology, 2012, 169(8): 807-815.
[10] Lovdal T, Lillo C.Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress[J]. Analytical biochemistry, 2009, 387(2): 238-242.
[11] De Carvalho K, Bespalhok Filho J C, Dos Santos T B, et al. Nitrogen starvation, salt and heat stress in coffee (Coffea arabica L.): Identification and validation of new genes for qPCR normalization[J]. Molecular Biotechnology, 2013, 53(3): 315-325.
[12] Xiao X L, Ma J B, Wang J R, et al.Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR[J]. Frontiers in Plant Science, 2015, 5: 788.
[13] 孙美莲, 王云生, 杨冬青, 等. 茶树实时荧光定量PCR分析中内参基因的选择[J]. 植物学报, 2010, 45: 579-587.
[14] Gohain B, Bandyopadhyay T, Borchetia S, et al.Identification and validation of stable reference genes in Camellia Species[J]. Journal of Biotechnology and Pharmaceutical Research, 2011, 2(1): 9-18.
[15] Gohain B, Bandyopadhyay T, Bhorali P, et al.Rubisco-bis-phosphate oxygenase (RuBP)-a potential housekeeping gene for qPCR assays in tea[J]. African Journal of Biotechnology, 2012, 11(51): 11193-11199.
[16] 郝姗. 茶树不同逆境条件下qRT-PCR适宜内参基因的筛选[D]. 南京: 南京农业大学, 2012: 47-55.
[17] Hao X, Horvath D, Chao W, et al.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)[J]. International Journal of Molecular Sciences, 2014, 15(12): 22155-22172.
[18] Okano K, Chutani K, Matsuo K.Suitable level of nitrogen fertilizer for tea (Camellia sinensis L.) plants in relation to growth, photosynthesis, nitrogen uptake and accumulation of free amino acids[J]. Japanese Journal of Crop Science, 1997, 66(2): 279-287.
[19] Urbanczyk-Wochniak E, Fernie A R.Metabolic profiling reveals altered nitrogen nutrient regimes have diverse effects on the metabolism of hydroponically-grown tomato (Solanum lycopersicum) plants[J]. Journal of Experimental Botany, 2005, 56(410): 309-321.
[20] 魏毅东, 陈玉, 郭海萍, 等. 水稻缺素胁迫下实时荧光定量RT-PCR的内参基因的选择[J]. 农业生物技术学报, 2013, 21: 1302-1312.
[21] Ruan J Y, Gerendas J, Hardter R, et al.Effect of nitrogen form and root-zone ph on growth and nitrogen uptake of tea (Camellia sinensis) plants[J]. Annals of Botany, 2007, 99(2): 301-310.
[22] Ruan J, Gerendás J, Härdter R, et al.Effect of root zone pH and form and concentration of nitrogen on accumulation of quality-related components in green tea[J]. Journal of the Science of Food and Agriculture, 2007, 87(8): 1505-1516.
[23] 蒋晓梅, 张新全, 严海东, 等. 柳枝稷根组织实时定量PCR分析中内参基因的选择[J]. 农业生物技术学报, 2014, 22: 55-63.
[24] Silver N, Best S, Jiang J, et al.Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR[J]. BMC Molecular Biology, 2006, 7(1): 33.
[25] Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [J]. Genome Biology, 2002, 3(7): research0034.1-0034.11.
[26] Andersen C L, Jensen J L, Orntoft T F.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 Research, 2004, 64(15): 5245-5250.
[27] 汪进, 添先凤, 江昌俊, 等. 茶树硝酸盐转运蛋白基因的克隆和表达分析[J]. 植物生理学报, 2014, 50: 983-988.
[28] Wang YY, Hsu PK, Tsay YF.Uptake, allocation and signaling of nitrate[J]. Trends in Plant Science, 2012, 17: 458-467.
[29] Wu X, Yang H, Qu C, et al. Sequence and expression analysis of the AMT gene family in poplar[J]. Frontiers in Plant Science, 2015, 6: 337.
[30] Faccioli P, Ciceri G P, Provero P, et al.A combined strategy of ′in silico′ transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies[J]. Plant Mol Biol, 2007, 63(5): 679-688.
[31] Olvera J, Wool I G.The primary structure of rat ribosomal protein L13[J]. Biochemical and Biophysical Research Communications, 1994, 201(1): 102-107.
[32] Martinez-Guitarte J L, Planello R, Morcillo G. Characterization and expression during development and under environmental stress of the genes encoding ribosomal proteins L11 and L13 in Chironomus riparius[J]. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology, 2007, 147(4): 590-596.
[33] Jain M, Tyagi S B, Thakur J K, et al.Molecular characterization of a light-responsive gene, breast basic conserved protein 1 (OsiBBC1), encoding nuclear-localized protein homologous to ribosomal protein L13 from Oryza sativa indica[J]. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2004, 1676(2): 182-192.
[34] Ye X, Zhang F M, Tao Y H, et al.Reference gene selection for quantitative real-time PCR normalization in different cherry genotypes, developmental stages and organs[J]. Scientia Horticulturae, 2015, 181: 182-188.
[35] Wang X C, Zhao Q Y, Ma C L, et al.Global transcriptome profiles of Camellia sinensis during cold acclimation[J]. BMC Genomics, 2013, 14(1): 415.
[36] Wei K, Wang L Y, Wu L Y, et al.Transcriptome analysis of indole-3-butyric acid-induced adventitious root formation in nodal cuttings of Camellia sinensis (L.)[J]. PLoS One, 2014, 9(9): e107201.
[37] Lim F-H, Fakhrana I N, Rasid O A, et al.Isolation and selection of reference genes for Ganoderma boninense gene expression study using quantitative Real-time PCR (qPCR)[J]. Journal of Oil Palm Research, 2014, 26(2): 170-181.
[38] An Y Q, Mcdowell J M, Huang S R, et al.Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues[J]. Plant Journal, 1996, 10(1): 107-121.
[39] 周晓惠, 刘军, 庄勇. 喀西茄内参基因实时荧光定量PCR表达稳定性评价[J]. 园艺学报, 2014, 41: 1731-1738.
[40] Czechowski T, Stitt M, Altmann T, et al.Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis[J]. Plant Physiol, 2005, 139(1): 5-17.
[41] Jain M.Genome-wide identification of novel internal control genes for normalization of gene expression during various stages of development in rice[J]. Plant Science, 2009, 176(5): 702-706.
[42] 周兰, 张利义, 张彩霞, 等. 苹果实时荧光定量PCR分析中内参基因的筛选[J]. 果树学报, 2012, 29(6): 965-970.
Options
文章导航

/

〈 〉

浙ICP备14034295号
浙公网安备 33019902000101号
版权所有 © 《茶叶科学》编辑部
技术支持：北京玛格泰克科技发展有限公司