Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2012, Vol. 38 ›› Issue (03): 471-478.doi: 10.3724/SP.J.1006.2012.00471


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 Online:2012-03-12 Published:2012-01-04
  • Contact: 刘红彦, E-mail: liuhy1219@163.com, Tel: 0371-65730166

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] WANG Xia, YIN Xiao-Yu, Yu Xiao-Ming, LIU Xiao-Dan. Effects of drought hardening on contemporary expression of drought stress memory genes and DNA methylation in promoter of B73 inbred progeny [J]. Acta Agronomica Sinica, 2022, 48(5): 1191-1198.
[2] LI Zeng-Qiang, DING Xin-Chao, LU Hai, HU Ya-Li, YUE Jiao, HUANG Zhen, MO Liang-Yu, CHEN Li, CHEN Tao, CHEN Peng. Physiological characteristics and DNA methylation analysis under lead stress in kenaf (Hibiscus cannabinus L.) [J]. Acta Agronomica Sinica, 2021, 47(6): 1031-1042.
[3] LU Hai, LI Zeng-Qiang, TANG Mei-Qiong, LUO Deng-Jie, CAO Shan, YUE Jiao, HU Ya-Li, HUANG Zhen, CHEN Tao, CHEN Peng. DNA methylation in response to cadmium stress and expression of different methylated genes in kenaf [J]. Acta Agronomica Sinica, 2021, 47(12): 2324-2334.
[4] Zuo-Min WANG,Jin LIU,Shi-Chao SUN,Xin-Yu ZHANG,Fei XUE,Yan-Jun LI,Jie SUN. Identification and Expression Analysis of Multidrug and Toxic Compound Extrusion Protein Family Genes in Colored Cotton [J]. Acta Agronomica Sinica, 2018, 44(9): 1380-1392.
[5] Wen-Juan YANG,Yan-Xin ZHANG,Lin-Hai WANG,Xin WEI,Dong-Hua LI,Yuan GAO,Pan LIU,Xiu-Rong ZHANG. Establishment of DNA Molecular Identification for A Sesame (Sesamum indicum L.) Applied Core Collection [J]. Acta Agronomica Sinica, 2018, 44(7): 1010-1020.
[6] LIU Hong-Yan, ZHOU Fang, LI Jun, YANG Min-Min, ZHOU Ting, HAO Guo-Cun,ZHAO Ying-Zhong . Anatomical Structure and Photosynthetic Characteristics of a Yellow Leaf Mutant YL1 in Sesame (Sesamum indicum L.) [J]. Acta Agron Sin, 2017, 43(12): 1856-1863.
[7] WU Kun1,2,WU Wen-Xiong1,YANG Min-Min1,LIU Hong-Yan1,HAO Guo-Cun1,ZHAO Ying-Zhong1,*. QTL Mapping for Oil, Protein and Sesamin Contents in Seeds of White Sesame [J]. Acta Agron Sin, 2017, 43(07): 1003-1011.
[8] SU Shi,LI Rui-Hang,LANG Dan-Ying,ZHANG Ke,HAO Xiao-Hu,LIU Yan,WANG Jun-Wei,XU Hong. Microstructure of Glandular Trichomes on Leaf Surface of Sesame and Changes of Trichome Secretions under Drought Condition [J]. Acta Agron Sin, 2016, 42(02): 278-294.
[9] Lü Gao-Qiang,WU Xiang-Yang,WANG Xin-Yu. Cloning and Characterization of a Novel Gene Encoding Proline-Rich Protein in Sesame [J]. Acta Agron Sin, 2015, 41(12): 1810-1818.
[10] WANG Zhen,FAN Xiao-Jing,ZHANG Miao,LI Xue-Yin,ZHANG Fang-Ning,LI Gui-Dong,SHEN Fang-Di,MA Ling-Jian*. Differential Expression of ATP Synthesis Related Gene in Fertility Conversion of Wheat BNS Male Sterile Line [J]. Acta Agron Sin, 2014, 40(08): 1501-1505.
[11] JIA Shuang-Wei,GAO Ying,ZHAO Kai-Jun. Cloning and Characterization of Brassica juncea Zinc Finger Protein Transcription Factor Gene Bj26 [J]. Acta Agron Sin, 2014, 40(07): 1174-1181.
[12] IN Heng,YU Qin-Yang,AN Li-Jia,LI Wen-Li. Cloning and Expression Analysis of an F-box Gene (SiFBX) Rapidly Responsive to Drought Stress [J]. Acta Agron Sin, 2014, 40(06): 1027-1034.
[13] HUANG Jie-Heng**,LI Wei**,QU Cun-Min,LIU Lie-Zhao,XU Xin-Fu,WANG Rui,LI Jia-Na*. Expression Characteristics of Key Genes in Lignin Pathway among Different Lodging Resistance Lines of Brassica napus L. [J]. Acta Agron Sin, 2013, 39(08): 1339-1344.
[14] LI Dong-Hua,LIU Wen-Ping,ZHANG Yan-Xin,WANG Lin-Hai,WEI Wen-Liang,GAO Yuan,DING Xia,WANG Lei,ZHANG Xiu-Rong. Identification Method of Drought Tolerance and Association Mapping for Sesame (Sesamum indicum L.) [J]. Acta Agron Sin, 2013, 39(08): 1425-1433.
[15] LIU Hong-Yan,WU Kun,YANG Min-Min,ZUO Yang,ZHAO Ying-Zhong. DNA Fingerprinting of Sesame (Sesamum indicum L.) Varieties (Lines) from Recent National Regional Trials in China [J]. Acta Agron Sin, 2012, 38(04): 596-605.
Full text



No Suggested Reading articles found!