A total of five traditional reference genes of Ustilaginoidea virens were systematically compared by using realtime quantitative PCR, including 18S rRNA, GAPDH, ubiquitin, βactin and αtubulin. The expression stability of the 5 candidate reference genes was evaluated with geNorm program when the fungus was 7, 8, 9 and 10 days old or exposed to 0.4 mol/L NaCl for 0 h，4 h， 8 h， 12 h， 16 h. The results showed that αtubulin2 and βactin were stably expressed when the fungus was at different days of age. For the case of 0.4 mol/L NaCl stress, βactin and αtubulin2 were also expressed stably. As a conclusion, βactin and αtubulin could be used as the reference genes for normalization of realtime quantitative PCR data.
GU Zhimin1,2, DING Zhengzhong1， CHEN Xifeng1， GUO Longbiao2， ZENG Dali2， QIAN Qian2,*， MA Bojun1,*
. Reference Gene Selection of Ustilaginoidea virens by Realtime PCR[J]. Chinese Journal OF Rice Science, 2012
, 26(5)
: 615
-618
.
DOI: 10.3969/j.issn.10017216.2012.05.015
\[1\]Schena M, Shalon D, Davis R W, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995, 270: 467470.
\[2\]Livak K J, Schmittgen T D. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method. Methods, 2001, 25: 402408.
\[3\]Vandesompele J, De Paepe A, Speleman F. Elimination of primerdimer artifacts and genomic coamplification using a twostep SYBR Green I realtime RTPCR. Anal Biochem, 2002, 303: 9598.
\[4\]Huggett J, Dheda K, Bustin S, et al. Realtime RTPCR normalization: Strategies and consideration. Genes & Immun， 2005, 6(4): 279284.
\[5\]Bustin S A. Quantification of mRNA using realtime reverse transcription PCR RTPCR: Trends and problems. J Mol Endocrinol, 2002, 29: 2339.
\[6\]Dheda K, Huggett J F, Bustin S A, et al. Validation of housekeeping genes for normalizing RNA expression in realtime PCR. Biotechniques, 2004, 37: 112119.
\[7\]Kim B R, Nam H Y, Kim S U, et al. Normalization of reverse transcription quantitativePCR with house keeping genes in rice. Biotechnol Letters, 2003, 25: 18691872.
\[8\]Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards: Use and limits. J Biotechnol, 1999, 75: 291295.
\[9\]Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of realtime quantitative RTPCR data by geometric averaging of multiple internal control genes. Genom Biol, 2002, 3(7): 112.
\[10\]Zhu H C, Xu X P, Xiao G Y, et al. Enhancing disease resistances of super hybrid rice with four antifungal genes. Sci China Ser C: Life Sci, 2007,50 (1): 3139.
\[11\]Kim K W, Park E W. Ultrastructure of spined conidia and hyphae of the rice false smut fungus Ustilaginoidea virens. Micron, 2007, 38: 626631.
\[12\]张君成, 张炳欣, 陈志谊, 等. 稻曲病菌分生孢子的生物学研究. 植物病理学报, 2003, 33(1): 4447.
\[13\]Vandesompele J, Preter D K, Pattyn F, et al. Accurate normalization of realtime quantitative RTPCR data by geometric averaging of multiple internal control genes. Genom Biol, 2002, 3(7): research 0034.10034.11.
\[14\]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. 植物学报, 2010, 45(5): 579587.
\[15\]Langer K, Ache P, Geiger D, et al. Poplar potassium transporters capable of controlling K+ homeostasis and K+ dependent xylogenesis. Plant J, 2002, 32(6): 9971009．
\[16\]Reid K E, Olsson N, Schlosser J, et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for realtime RTPCR during berry development. BMC Plant Biol, 2006, 6: 27．
\[17\]Li L, Xu J, Xu Z H, et al. Brassinosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis. Plant Cell, 2005, 17(10): 27382753．
\[18\]吴文凯, 刘成前, 周志刚, 等. 用于莱茵衣藻荧光定量PCR分析的内参基因选择. 植物生理学通讯, 2009, 45(7): 667672.
\[19\]阙友雄, 许莉萍, 徐景升, 等. 甘蔗基因表达定量 PCR 分析中内参基因的选择. 热带作物学报, 2009, 30(3): 274278.
\[20\]张积森, 李伟, 阙友雄, 等. 斑茅两个看家基因片段的克隆及其在基因芯片中的应用. 热带亚热带植物学报, 2007, 15 (4): 277283.
