欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2012, Vol. 38 ›› Issue (03): 471-478.doi: 10.3724/SP.J.1006.2012.00471

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

茎点枯病菌诱导下芝麻内参基因的筛选

刘莉铭1,2,3,刘红彦1,2,3,*,田保明1   

  1. 1 郑州大学生物工程系，河南郑州 450001；2 河南省农业科学院植物保护研究所，河南郑州 450002；3 河南省农作物病虫害防治重点实验室，河南郑州 450002
  • 收稿日期:2011-09-19 修回日期:2011-12-15 出版日期:2012-03-12 网络出版日期:2012-01-04
  • 通讯作者: 刘红彦, E-mail: liuhy1219@163.com, Tel: 0371-65730166
  • 基金资助:

    本研究由现代农业产业技术体系建设专项资金(CARS-15-1-05)资助。

Selection of Reference Genes from Sesame Infected by Macrophomina phaseolina

LIU Li-Ming1,2,3,LIU Hong-Yan1,2,3,*,TIAN Bao-Ming1   

  1. 1 Bioengineering Department, Zhengzhou University, Zhengzhou 450001, China; 2 Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; 3 Henan Key Laboratory for Control of Crop Diseases and Insect Pests, Zhengzhou 450002, China
  • Received:2011-09-19 Revised:2011-12-15 Published:2012-03-12 Published online:2012-01-04
  • Contact: 刘红彦, E-mail: liuhy1219@163.com, Tel: 0371-65730166

PDF

1357

被引次数

15

摘要： 用CodeHop方法设计简并引物克隆得到了GAPDH、β-actin、α-tubulin、UBQ5、RPL4、eIF4A和eEF1α 7个看家基因的部分序列，将这7个基因和GenBank中已公布的18S rRNA、NADHD 2个基因共9个基因作为候选内参基因，利用实时荧光定量PCR技术，分析其在茎点枯病菌诱导下芝麻中的表达稳定性。经BestKeeper、NormFinder和GeNorm软件分析可知，UBQ5、eIF4A、α-tubulin 3个基因表达均较稳定，eEF1α变化最大。当使用多基因作为内参基因时，选择这3个最稳定的候选内参基因即可准确矫正定量结果。

关键词: 芝麻, 茎点枯病菌, qRT-PCR, 内参基因

Abstract: Stem rot caused by Macrophomina phaseolina (Tassi) Goid. is the most important disease in sesame, worldwide, the use of sesame cultivars resistant to stem rot provides an effective, economical, and environmentally friendly method to control the disease. The study on sesame defense response plays important roles in breeding resistant cultivars, andqRT-PCRwas an important method in studying plant defense response. In order to select the proper reference genes which can normalize expression of the target gene in sesame induced by Macrophomina phaseolina, nine genes 18S rRNA, NADHD, GAPDH, β-actin, α-tubulin, UBQ5, RPL4, eIF4A, and eEF1α, was analyzed by qRT-PCR. Among the nine genes, last seven were designed using CodeHop. BestKeeper, NormFinder and GeNorm analyses showed that UBQ5, eIF4A and α-tubulin were the top three optimum choices while eEF1α varied greatly. The analysis revealed that the three most stable genes UBQ5, eIF4A, α-tubulin were enough to obtain an accurate result when using multiple genes as control.

Key words: Sesame, Macrophomina phaseolina, qRT-PCR, Reference gene

[1]Guo Y(郭杨), Chen S-J(陈世界), Guo W-Z(郭万柱), Li J(李璟). Advance in fluorescent quantitative PCR and its applications. Prog Vet Med (动物医学进展), 2009, 30(2): 78–82 (in Chinese with English abstract)

[2]Suzuki T, Higgins P J, Crawford D. Control selection for RNA quantitation. Biotechniques, 2000, 29: 332–337

[3]Sun M-L(孙美莲), Wang Y-S(王云生), Yang D-Q(杨冬青), Wei C-L(韦朝领), Gao L-P(高丽萍), Xia T(夏涛), Shan Y(单育), Luo Y(骆洋). Reference genes for real-time fluorescence quantitative PCR in Camellia sinensis. Chin Bull Bot (植物学报), 2010, 45(5): 579–587 (in Chinese with English abstract)

[4]Barsalobres-Cavallari C F, Severino F E, Maluf M P, Maia I G. Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Mol Biol, 2009, 10: 1–10

[5]Jain M, Nijhawan A, Tyagi A K, Khurana J P. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun, 2006, 345: 646–651

[6]Luo H, Chen S, Wan H, Chen F, Gu C, Liu Z. Candidate reference genes for gene expression studies in water lily. Anal Biochem, 2010, 404: 100–102

[7]Maroufi A, Van B E, De L M. Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol, 2010, 11: 1–12

[8]Li Q-F(李钱峰), Jiang M-Y(蒋美艳), Yu H-X(于恒秀), Xin S-W(辛世文), Gu M-H(顾铭洪), Liu Q-Q(刘巧泉). Selection of internal reference genes for quantitative RT-PCR analysis of total RNA from endosperm of rice (Oryza sativa L.). J Yangzhou Univ (Agric Life Sci Edn) (扬州大学学报?农业与生命科学版), 2008, 29(2): 61–66 (in Chinese with English abstract)

[9]Nicot N, Hausman J F, Hoffmann L, Evers D. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot, 2005, 56: 2907–2914

[10]Andersen C L, Jensen J L, Ørntoft 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. Cancer Res, 2004, 64: 5245–5250

[11]Xie T-D(谢天丁), Pei G-Y(裴桂英), Liu B-C(刘保才), Ma S-F(马赛飞), Liu J(刘健). Control effect experiment of Sesame Fusarium Wilt and Stem Blight. J Hebei Agric Sci (河北农业科学), 2010, 14(4): 60–61 (in Chinese with English abstract)

[12]Huang J(黄菁), Wang S-L(王少丽), Qiao C-L(乔传令). Automated programming of degenerate primers and the cloning of the diamondback esterase gene. Entomolog Knowl (昆虫知识), 2002, 39(6): 458–461 (in Chinese with English abstract)

[13]Rose T M, Schultz E R, Henikoff J G, Pietrokovski S, McCallum C M, Henikoff S. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly-related sequences. Nucl Acids Res, 1998, 26: 1628–1635

[14]Rose T M, Henikoff J G, Henikoff S. CODEHOP (consensus-degenerate hybrid oligonucleotide primer) PCR primer design. Nucl Acids Res, 2003, 31: 3763–3766

[15]Yu H-S(于寒松), Zhang J-X(张继星), Li Y-F(李彦舫), Ma L-Q(马兰青), Hu Y-H(胡耀辉). Full-length cDNA cloning of glycosyltransferase family from Rhodiola sachalinensis. Food Sci (食品科学), 2010, 31(21): 244–247 (in Chinese with English abstract)

[16]Chen C-F(陈彩芳), Wen H-S(温海深), He F(何峰), Dong S-L(董双林). Programming design of degenerate primers and cloning half-smooth tongue sole cynoglossus semila evis CYP17 gene. Period Ocean Univ China (中国海洋学报), 2009, 39(6): 1213–1218 (in Chinese with English abstract)

[17]Provencher C, Lapointe G, Sirois S, Vancalsteren M R, Roy D. Consensus-degenerate hybrid oligonucleotide primers for ampli?cation of priming glycosyl transferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group. Appl Environ Microbiol, 2003, 69: 3299–3307

[18]Lindqvist N, Lönngren U, Agudo M, Näpänkangas U, Vidal-Sanz M, Hallböök F. Multiple receptor tyrosine kinases are expressed in adult rat retinal ganglion cells as revealed by single-cell degenerate primer polymerase chain reaction. Ups J Med Sci, 2010, 115: 65–80

[19]Hu R-B(胡瑞波), Fan C-M(范成明), Fu Y-F(傅永福). Reference gene selection in plant real-time quantitative reverse transcription PCR (qRT-PCR). J Agric Sci Technol (中国农业科技导报), 2009, 11(6): 30–36 (in Chinese with English abstract)

[20]Dong X-L(董晓丽), Wang J-Q(王加启), Bu D-P(卜登攀), Zhang C-L(张春林), Li S-S(李珊珊), Zhao G-Q(赵国琦). Research advancement of reference gene in the fluorescent quantitative PCR and its application. China Animal Husb Vet Med (中国畜牧兽医), 2009, 36(9): 83–85 (in Chinese)

[21]Lee P D, Sladek R, Greenwood C M T, Hudson T J. Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res, 2002, 12: 292–297

[22]Pfaffl M W, Tichopad A, Prgomet C, Neuvians T P. Determination of stable housekeeping genes, differentially regulated tar-get genes and sample integrity: bestkeeper-excel-based tool using pair-wise correlations. Biotechnol Lett, 2004, 26: 509–515

[23]Vandesompele J, De P K, Pattyn F, Poppe B, Van R N, De P A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 2002, 3: 1–13

[24]Akilesh S, Shafer D J, Roopenian D. Customized molecular phenotyping by quantitative gene expression and pattern recognition analysis. Genome Res, 2003, 13: 1719–1727

[25]Brunner A M, Yakovlev I A, Strauss S H. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol, 2004, 4: 1–7

[26]Wu W-K(吴文凯), Liu C-Q(刘成前), Zhou Z-G(周志刚), Lu S(卢山). The selection of reference genes in Chlamydomonas reinhardtii P A dangeard by real-time quantitative PCR. Plant Physiol Commun (植物生理学通讯), 2009, 45(7): 667–672 (in Chinese with English abstract)

[27]Li M(李梅), Guo J-H(郭建华), Liu X-D(刘学东), Lü Y-C(吕彦超), Wang W-Y(王文艳). Comparison of inoculation method for anthracnose resistance in bean seedling. Tianjing Agric Sci (天津农业科学), 2009, 15(1): 31–32 (in Chinese with English abstract)
[1] 王霞, 尹晓雨, 于晓明, 刘晓丹. 干旱锻炼对B73自交后代当代干旱胁迫记忆基因表达及其启动子区DNA甲基化的影响[J]. 作物学报, 2022, 48(5): 1191-1198.
[2] 李增强, 丁鑫超, 卢海, 胡亚丽, 岳娇, 黄震, 莫良玉, 陈立, 陈涛, 陈鹏. 铅胁迫下红麻生理特性及DNA甲基化分析[J]. 作物学报, 2021, 47(6): 1031-1042.
[3] 卢海, 李增强, 唐美琼, 罗登杰, 曹珊, 岳娇, 胡亚丽, 黄震, 陈涛, 陈鹏. 红麻DNA甲基化响应镉胁迫及甲基化差异基因的表达分析[J]. 作物学报, 2021, 47(12): 2324-2334.
[4] 杨文娟,张艳欣,王林海,魏鑫,黎冬华,高媛,刘盼,张秀荣. 一个芝麻应用核心种质的DNA分子身份证构建[J]. 作物学报, 2018, 44(7): 1010-1020.
[5] 刘红艳,周芳,李俊,杨敏敏,周婷,郝国存,赵应忠. 芝麻黄化突变体YL1的叶片解剖学及光合特性[J]. 作物学报, 2017, 43(12): 1856-1863.
[6] 吴坤,吴文雄,杨敏敏,刘红艳,郝国存,赵应忠. 白芝麻籽粒油脂、蛋白质及芝麻素含量QTL定位分析[J]. 作物学报, 2017, 43(07): 1003-1011.
[7] 苏适，李瑞航，郎丹莹，张柯，郝小虎，刘研，王军卫，徐虹. 芝麻叶腺毛显微结构及干旱条件下腺毛分泌物的变化[J]. 作物学报, 2016, 42(02): 278-294.
[8] 吕高强，吴向阳，王心宇. 芝麻中一个富含脯氨酸新基因的克隆与特征分析[J]. 作物学报, 2015, 41(12): 1810-1818.
[9] 黎冬华,刘文萍,张艳欣,王林海,危文亮,高媛,丁霞,王蕾,张秀荣. 芝麻耐旱性的鉴定方法及关联分析[J]. 作物学报, 2013, 39(08): 1425-1433.
[10] 岳文娣，魏利斌，张体德，李春，苗红梅，张海洋. 芝麻种质资源SSR标记遗传多样性与群体结构[J]. 作物学报, 2012, 38(12): 2286-2296.
[11] 刘红艳，吴坤，杨敏敏，左阳，赵应忠. 国家芝麻区域试验新品种(系)的DNA指纹分析[J]. 作物学报, 2012, 38(04): 596-605.
[12] 张岗，董艳玲，夏宁，张毅，王晓杰，屈志鹏，李依民，黄丽丽，康振生. 利用cDNA-AFLP技术分析小麦成株抗条锈性差异基因表达特征[J]. 作物学报, 2010, 36(3): 401-409.
[13] 车卓，张艳欣，孙建，张秀荣，尚勋武，王化俊. 应用SRAP标记分析黑芝麻核心种质遗传多样性[J]. 作物学报, 2009, 35(10): 1936-1941.
[14] 魏利斌;张海洋;郑永战;郭旺珍;张天真. 芝麻EST-SSR标记的开发和初步研究[J]. 作物学报, 2008, 34(12): 2077-2084.
[15] 杨晓丽;张海洋;郭旺珍;郑永战;苗红梅;魏利斌;张天真. 芝麻核雄性不育系ms86-1小孢子败育过程的超微结构[J]. 作物学报, 2008, 34(11): 1894-1900.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2