实时荧光定量PCR筛选稻曲病菌内参基因

doi:10.3969/j.issn.10017216.2012.05.015

中国水稻科学 ›› 2012, Vol. 26 ›› Issue (5): 615-618.DOI: 10.3969/j.issn.10017216.2012.05.015

实时荧光定量PCR筛选稻曲病菌内参基因

顾志敏^1,2,丁正中¹,陈析丰¹,郭龙彪² ,曾大力² ,钱前^2,* ,马伯军^1,*

¹ 浙江师范大学化学与生命科学学院，浙江金华 321004；² 中国水稻研究所水稻生物学国家重点实验室，浙江杭州310006；

收稿日期:2012-01-16 修回日期:2012-03-12 出版日期:2012-09-10 发布日期:2012-09-10
通讯作者: 钱前2,* ,马伯军1,*
基金资助:
国家自然科学基金资助项目（31171519， 31101130）；浙江省自然科学基金资助项目（LY12CD6001）。

Reference Gene Selection of Ustilaginoidea virens by Realtime PCR

GU Zhimin ^1,2, DING Zhengzhong ¹， CHEN Xifeng ¹， GUO Longbiao ²， ZENG Dali ² ， QIAN Qian ^2,* ， MA Bojun ^1,*

¹ College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China; ²State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China;

Received:2012-01-16 Revised:2012-03-12 Online:2012-09-10 Published:2012-09-10
Contact: QIAN Qian2,*， MA Bojun1,*

摘要/Abstract

摘要： 通过实时定量PCR分析了稻曲病菌中5个传统内参基因18S rRNA、GAPDH、ubiquitin、βactin、αtubulin的mRNA差异表达情况，并利用geNorm软件分析了它们在4个发育时期和NaCl胁迫处理中的表达稳定性。结果表明，在菌龄为7 d、8 d、9 d、10 d的稻曲病菌中，αtubulin2和βactin表达稳定；在0.4 mol/L NaCl溶液处理0 h、4 h、8 h、12 h、16 h后，βactin和αtubulin2表达稳定。因此，当利用荧光定量PCR分析比较稻曲病菌基因表达差异时，可选择αtubulin和βactin作为内参基因。

关键词: 稻曲病菌, 实时定量PCR, 内参基因, geNorm软件

Abstract: A total of five traditional reference genes of Ustilaginoidea virens were systematically compared by using realtime quantitative PCR, including 18S rRNA, GAPDH, ubiquitin, βactin and αtubulin. 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 αtubulin2 and βactin were stably expressed when the fungus was at different days of age. For the case of 0.4 mol/L NaCl stress, βactin and αtubulin2 were also expressed stably. As a conclusion, βactin and αtubulin could be used as the reference genes for normalization of realtime quantitative PCR data.

Key words: Ustilaginoidea virens, realtime quantitative PCR, reference gene, geNorm program

中图分类号:

顾志敏1,2,丁正中1,陈析丰1,郭龙彪2 ,曾大力2 ,钱前2,* ,马伯军1,* . 实时荧光定量PCR筛选稻曲病菌内参基因[J]. 中国水稻科学, 2012, 26(5): 615-618.

GU Zhimin1,2, DING Zhengzhong1， CHEN Xifeng1， GUO Longbiao2， ZENG Dali2， QIAN Qian2,*， MA Bojun1,*. Reference Gene Selection of Ustilaginoidea virens by Realtime PCR[J]. Chinese Journal of Rice Science, 2012, 26(5): 615-618.

参考文献

\[1\]Schena M, Shalon D, Davis R W, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995, 270: 467470.

\[2\]Livak K J, Schmittgen T D. Analysis of relative gene expression data using realtime quantitative PCR and the 2-ΔΔCT method. Methods, 2001, 25: 402408.

\[3\]Vandesompele J, De Paepe A, Speleman F. Elimination of primerdimer artifacts and genomic coamplification using a twostep SYBR Green I realtime RTPCR. Anal Biochem, 2002, 303: 9598.

\[4\]Huggett J, Dheda K, Bustin S, et al. Realtime RTPCR normalization: Strategies and consideration. Genes & Immun， 2005, 6(4): 279284.

\[5\]Bustin S A. Quantification of mRNA using realtime reverse transcription PCR RTPCR: Trends and problems. J Mol Endocrinol, 2002, 29: 2339.

\[6\]Dheda K, Huggett J F, Bustin S A, et al. Validation of housekeeping genes for normalizing RNA expression in realtime PCR. Biotechniques, 2004, 37: 112119.

\[7\]Kim B R, Nam H Y, Kim S U, et al. Normalization of reverse transcription quantitativePCR with house keeping genes in rice. Biotechnol Letters, 2003, 25: 18691872.

\[8\]Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards: Use and limits. J Biotechnol, 1999, 75: 291295.

\[9\]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. Genom Biol, 2002, 3(7): 112.

\[10\]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): 3139.

\[11\]Kim K W, Park E W. Ultrastructure of spined conidia and hyphae of the rice false smut fungus Ustilaginoidea virens. Micron, 2007, 38: 626631.

\[12\]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): 4447.

\[13\]Vandesompele J, Preter D K, Pattyn F, et al. Accurate normalization of realtime quantitative RTPCR data by geometric averaging of multiple internal control genes. Genom Biol, 2002, 3(7): research 0034.10034.11.

\[14\]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): 579587.

\[15\]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): 9971009．

\[16\]Reid K E, Olsson N, Schlosser J, et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for realtime RTPCR 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): 27382753．

\[18\]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): 667672.

\[19\]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): 274278.

\[20\]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): 277283.

[1]	伏荣桃, 陈诚, 王剑, 赵黎宇, 陈雪娟, 卢代华. 转录组和代谢组联合分析揭示稻曲病菌的致病因子[J]. 中国水稻科学, 2024, 38(4): 375-385.
[2]	尹潇潇, 张芷菡, 颜绣莲, 廖蓉, 杨思葭, 郭岱铭, 樊晶, 赵志学, 王文明. 多个稻曲病菌效应因子的信号肽验证和表达分析[J]. 中国水稻科学, 2024, 38(3): 256-265.
[3]	伏荣桃, 王剑, 陈诚, 赵黎宇, 陈雪娟, 卢代华. 水稻幼穗响应稻曲病菌毒素胁迫早期的转录组分析[J]. 中国水稻科学, 2022, 36(5): 447-458.
[4]	徐红星, 王国荣, 鲁艳辉, 杨亚军, 郑许松, 田俊策, 吕仲贤. 二化螟实时荧光定量PCR内参基因筛选和表达稳定性评价[J]. 中国水稻科学, 2019, 33(1): 75-84.
[5]	丁慧, 俞咪娜, 王亚会, 于俊杰, 尹小乐, 薄惠文, 黄星, 刘永锋. 稻曲病菌T-DNA插入突变体B2510的插入位点分析[J]. 中国水稻科学, 2016, 30(5): 541-551.
[6]	王亚会, 刘永锋, 陆凡, 俞咪娜, 黄磊, 郑梦婷, 于俊杰, 尹小乐. 稻曲病菌T-DNA插入突变体B1464插入位点分析[J]. 中国水稻科学, 2015, 29(3): 311-318.
[7]	饶玉春1,2,#,丁正中1,3,#,陈析丰1,曾大力2,马伯军1,顾志敏1,* . 稻曲病菌UvHog1基因的克隆及表达分析[J]. 中国水稻科学, 2014, 28(1): 9-14.
[8]	郑静1，2 ,张震2，* ,姜华2 ,王艳丽2 ,柴荣耀2 ,邱海萍2 ,毛雪琴2 ,王教瑜2 ,杜新法2 ,赖朝晖3 ,孙国昌2，*. 稻曲病菌分生孢子实时PCR定量检测方法的建立及初步应用[J]. 中国水稻科学, 2012, 26(4): 500-505.
[9]	王娜1,任佐华1,邓林伟2,陈娟芳3 ,刘二明1，*. 稻曲病菌休眠与非休眠厚垣孢子胞壁多糖结构与组分[J]. 中国水稻科学, 2012, 26(3): 356-360.
[10]	祭芳,曹欢,徐剑宏,殷宪超,史建荣*. 高效液相色谱串联质谱法定量检测稻谷中的稻曲病菌毒素A和D[J]. 中国水稻科学, 2012, 26(2): 246-250.
[11]	刘连盟,王玲,黄雯雯,刘恩勇,黄世文. 水稻稻曲病菌G蛋白β亚基基因的克隆、表达与序列分析[J]. 中国水稻科学, 2010, 24(4): 353-359 .
[12]	张震,杜新法,柴荣耀,毛雪琴,邱海萍,王艳丽,王教瑜,孙国昌. 根癌农杆菌介导遗传转化稻曲病菌[J]. 中国水稻科学, 2006, 20(4): 440-442 .
[13]	张君成, 张炳欣, 陈志谊, 刘永锋, 陆凡. 稻曲病的接种方法及其效果初探[J]. 中国水稻科学, 2003, 17(4): 390-392 .
[14]	周永力,章琦. 稻曲病菌分离技术的初探[J]. 中国水稻科学, 1999, 13(3): 186-188 .

实时荧光定量PCR筛选稻曲病菌内参基因

Reference Gene Selection of Ustilaginoidea virens by Realtime PCR

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 14

编辑推荐

Metrics