不同非生物胁迫下香樟实时定量PCR内参基因的选择

doi:10.13560/j.cnki.biotech.bull.1985.2016.10.025

摘要/Abstract

摘要： 旨在获得香樟（Cinnamomum camphora L.）在不同非生物胁迫下表达稳定的内参基因,以香樟实生苗为材料,利用实时定量PCR（quantitative real-time polymerase chain reaction,qRT-PCR）技术研究分析了两个香樟候选内参基因CcACTc和CcEF1α在不同非生物胁迫下的表达量,并通过BestKeeper软件对CcACTc和CcEF1α的稳定性进行了分析。结果表明,在低温处理中,CcACTc的稳定性较好,在干旱、高盐和ABA处理中则CcEF1α的稳定性较好,并且其在低温处理中也相对稳定。由此可知,CcEF1α更适合作为内参基因应用于香樟非生物胁迫相关基因的定量分析。

关键词: 香樟, qRT-PCR, 内参基因, 非生物胁迫

Abstract: This study is to obtain the optimal reference genes of Cinnamomum camphora under different abiotic stresses. The expression level and expression stability of two candidate reference genes CcACTc and CcEF1α in C. camphora under various abiotic stresses were analyzed using quantitative real-time polymerase chain reaction（qRT-PCR）technique and BestKeeper software,respectively. The results indicated that,CcEF1α was relatively stable under low temperature treatment,and performed higher stability under other abiotic stress,such as drought,high salinity and ABA treatment compared to CcEF1α. Accordingly,CcEF1α is a considerable reference gene for quantitative analysis of stress-related genes in C. camphora.

Key words: Cinnamomum camphora, qRT-PCR, reference gene, abiotic stress

张佳佳, 张力维, 李勇鹏, 焦晓琳, 朱逢玲, 杜丽. 不同非生物胁迫下香樟实时定量PCR内参基因的选择[J]. 生物技术通报, 2016, 32(10): 205-211.

ZHANG Jia-jia, ZHANG Li-wei, LI Yong-peng, JIAO Xiao-lin, ZHU Feng-ling, DU Li. Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Cinnamomum camphora Under Various Abiotic Stresses[J]. Biotechnology Bulletin, 2016, 32(10): 205-211.

参考文献

[1] Le DT, Aldrich DL, Valliyodan B, et al. Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions[J]. PLoS One, 2012, 7（9）：1602-1603.
[2] Gachon C, Mingam A, Charrier B. Real-time PCR：what relevance to plant studies[J]. Journal of Experimental Botany, 2004, 55（402）：1445-1454.
[3] Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR[J]. Nature Protocols, 2006, 1（3）：1559-1582.
[4] Hu RB, Fan CM, Li HY, et al. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR[J]. BMC Molecular Biology, 2009, 10（1）：93.
[5] Caldana C, Scheible WR, Mueller-Roeber B, et al. A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors[J]. Plant Methods, 2007, 3（1）：7.
[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] Bjarnadottir H, Jonsson JJ. A rapid real-time qRT-PCR assay for ovine β-actin mRNA[J]. Journal of Biotechnology, 2005（2）：173-182.
[8] 牙库甫江· 阿西木, 关波, 张富春. 植物基因表达转录分析中内参基因的选择与应用[J]. 生物技术通报, 2011（7）：7-11.
[9] 张玉芳, 赵丽娟, 曾幼玲. 基因表达研究中内参基因的选择与应用[J]. 植物生理学报, 2014, 50（8）：1119-1125.
[10] Nicot N, Hausman JF, Hoffmann L, et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 2005, 56（421）：2907-2914.
[11] Lopez-Pardo R, Ruiz de Galarreta JI, Ritter E. Selection of housekeeping genes for qRT-PCR analysis in potato tubers under cold stress[J]. Molecular Breeding, 2013, 31（1）：39-45.
[12] Bustin SA, Gyselman VG, Siddiqi S, et al. Cytokeratin 20 is not atissue specific marker for the detection of malignant epithelial cells in the blood of colorectal cancer patients[J]. International Journal of Surgical Investigation, 2000, 2（1）：49-57.
[13] Wan HJ, Zhao ZJ, Qian CT, et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry, 2010, 399（2）：257-261.
[14] Chen Y, Hu B, Tan Z, et al. Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses[J]. Plant Cell Reports, 2015, 34（10）：1825-1834.
[15] 韩晓雪, 韩佳轩, 姜晶. 番茄在非生物胁迫下实时定量RT-PCR中内参基因的筛选[J]. 分子植物育种, 2015, 13（4）：822-831.
[16] 李勇鹏, 张力维, 姚瑶, 等. 香樟Actin基因的克隆及表达分析[J]. 生物技术通报, 2015, 31（5）：120-127.
[17] Lin LY, Han XJ, Chen YC, et al. Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba[J]. Mgg Molecular & General Genetics, 2013, 288（12）：727-737.
[18] 张力维, 李勇鹏, 姚瑶, 等. 香樟延伸因子EF1α基因片段的克隆和表达分析[J]. 中南林业科技大学学报, 2015, 35（5）：122-128.
[19] Pfaffl MW, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity：bestkeeper-excel-based tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26（6）：509-515.
[20] Mehta R, Birerdinc A, Hossain N, et al. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples[J]. BMC Molecular Biology, 2010, 11（39）：1471-2199.
[21] 苏晓娟, 樊保国, 袁丽钗, 等. 实时荧光定量PCR分析中毛果杨内参基因的筛选和验证[J]. 植物学报, 2013, 48（5）：507-518.
[22] Bustin SA, Benes V, Nolan T, et al. Quantitative real-time RT-PCR-a perspective[J]. Journal of Molecular Endocrinology, 2005, 34（3）：597-601.
[23] Santella L, Chun JT. Actin, more than just a house-keeping protein at the scene of fertilization[J]. Science China Life Sciences, 2011, 54（8）：733-743.
[24] 周冰, 曹诚, 刘传暄. 翻译延伸因子1A的研究进展[J]. 生物技术通讯, 2007, 18（2）：281-284.
[25] Wang XM, Dong J, Liu Y, et al. A novel dehydration-responsive element-binding protein from Caragana korshinskii is involved in the response to multiple abiotic stresses and enhances stress tolerance in transgenic tobacco[J]. Plant Molecular Biology Reporter, 2010, 28（4）：664-675.
[26] Zhou ML, Ma JT, Zhao YM, et al. Improvement of drought and salt tolerance in Arabidopsis and Lotus corniculatus by overexpression of a novel DREB transcription factor from Populus euphratica[J]. Gene, 2012, 506（1）：10-7.
[27] Wu DY, Ji J, Wang G, et al. LchERF, a novel ethylene- responsive transcription factor from Lycium chinense, confers salt tolerance in transgenic tobacco[J]. Plant Cell Reports, 2014, 33（12）：2033-2045.
[28] Wang LH, Gao JH, Qin XB, et al. JcCBF2 gene from Jatropha curcas improves freezing tolerance of Arabidopsis thaliana during the early stage of stress[J]. Molecular Biology Reports, 2015, 42（5）：937-945.
[29] Bouaziz D, Jbir R, Charfeddine S, et al. The StDREB1 transcription factor is involved in oxidative stressresponse and enhances tolerance to salt stress[J]. Plant Cell Tiss Organ Cult, 2014, 121（1）：237-248.
[30] Guo C, Zhang JQ, Peng T, et al. Structural and expression analyses of three PmCBFs from Prunus mume[J]. Biologia Plantarum, 2014, 58（2）：247-255.
[31] Kudo K, Oi T, Uno Y. Functional characterization and expression profiling of a DREB2-type gene from lettuce（Lactuca sativa L.） [J]. Plant Cell Tiss Organ Cult, 2014, 116（1）：97-109.
[32] Yang Q, Yin JJ, Li G, et al. Reference gene selection for qRT-PCR in Caragana korshinskii Kom. under different stress conditions[J]. Molecular Biology Reports, 2014, 41（4）：2325-2334.
[33] Huang L, Yan H, Jiang X, et al. Evaluation of candidate reference genes for normalization of quantitative RT-PCR in switchgrass under various abiotic stress conditions[J]. Bioenergy Research, 2014, 7（4）：1201-1211.
[34] Wang Z, Chen Y, Fang H, et al. Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions[J]. Molecular Genetics and Genomics, 2014, 289（5）：1023-1035.
[35] Jiang Q, Wang F, Li MY, et al. Selection of suitable reference genes for qPCR normalization under abiotic stresses in Oenanthe javanica（BI. ）DC[J]. PLoS One, 2014, 9（3）：e92262.
[36] Hong SY, Seo PJ, Yang MS, et al. Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR[J]. BMC Plant Biology, 2008, 8（1）：112.