大豆胞囊线虫侵染下野生大豆根组织实时定量PCR分析中内参基因的筛选
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摘要
以高抗和高感HG 0大豆胞囊线虫的野生大豆种质为试验材料,采用q RT-PCR技术检测了ACT11(Glyma.18G290800)、Tubulin-motif(Glyma.19G194800)等24个候选持家基因在不同大豆胞囊线虫侵染时期(接种后9,15和20 d)野生大豆根系中的基因表达情况,除Actin(Glyma.08G182200)出现了非特异扩增外,其它23个持家基因的q RT-PCR扩增效果均理想,扩增特异性高。分析上述23个持家基因的表达丰度,并利用Best Keeper、ge Norm和Normfinder对其表达稳定性进行评价。结果表明:持家基因间的表达丰度不尽相同,而且有些持家基因在不同品种间或不同处理间(接种虫卵和接种水)也存在表达丰度的差异。23个持家基因的表达稳定性也不相同,综合来看,表达最不稳定的基因为Cons9(Glyma.10G152200)、Cons2(Glyma.17G138500)、Cons1(Glyma.15G270900)和Tubulin-motif(Glyma.20G136000),本试验条件下它们不适宜作内参基因;其余持家基因相对稳定,本试验条件下可选择Tubulinmotif(Glyma.15G132200),Cons6/SKIP16(Glyma.12G051100)或Tubulin-motif(Glyma.05G110200)作为内参基因,它们表达量较高,表达最为稳定,其Ct平均值分别为25.7、26.5和25.0,标准偏差分别为0.540、0.575和0.490,ge Norm软件评价其稳定性的M值分别为0.698、0.715和0.727。该研究为野生大豆抗胞囊线虫相关基因的表达分析提供了参考。
Real-time quantitative PCR( q RT-PCR) has been widely used in crops for gene expression analysis,and reference genes are required in q RT-PCR analysis to minimize influences of RNA quality and quantity and efficiency of reverse transcription. Housekeeping genes are often selected as reference genes,however many of the housekeeping genes provide stable expression under only certain environments. So it is necessary to identify and select suitable reference genes for a given experiment. In our study,24 candidate housekeeping genes were selected for their gene expression analysis,there happened nonspecific amplification products for q RT-PCR of Actin( Glyma. 08 G182200). The other 23 candidate housekeeping genes were evaluated for their expression level and stability in roots of susceptible and resistant wild soybean accessions under soybean cyst nematode HG 0 infection at 9,15 and 20 days after inoculation. Housekeeping genes had different expression level,and some of them had different expression level between wild soybean accessions,or between different treatments. There also existed difference in expression stability among the 23 housekeeping genes. As a whole,Cons9( Glyma. 10 G152200),Cons2( Glyma. 17 G138500),Cons1( Glyma. 15 G270900) and Tubulin-motif( Glyma. 20 G136000) were the top four least stable genes,and they were not suitable for the study. The three housekeeping genes of Tubulin-motif( Glyma. 15 G132200),Cons6/SKIP16( Glyma. 12 G051100) and Tubulin-motif( Glyma. 05 G110200) were the most stable genes with M value of 0. 698,0. 715 and 0. 727 respectively and with their expression level of Ct = 25. 7,26. 5 and 25. 0 respectively,which indicated that they are ideal reference genes for q RT-PCR analysis under our experiment conditions.
Real-time quantitative PCR( q RT-PCR) has been widely used in crops for gene expression analysis,and reference genes are required in q RT-PCR analysis to minimize influences of RNA quality and quantity and efficiency of reverse transcription. Housekeeping genes are often selected as reference genes,however many of the housekeeping genes provide stable expression under only certain environments. So it is necessary to identify and select suitable reference genes for a given experiment. In our study,24 candidate housekeeping genes were selected for their gene expression analysis,there happened nonspecific amplification products for q RT-PCR of Actin( Glyma. 08 G182200). The other 23 candidate housekeeping genes were evaluated for their expression level and stability in roots of susceptible and resistant wild soybean accessions under soybean cyst nematode HG 0 infection at 9,15 and 20 days after inoculation. Housekeeping genes had different expression level,and some of them had different expression level between wild soybean accessions,or between different treatments. There also existed difference in expression stability among the 23 housekeeping genes. As a whole,Cons9( Glyma. 10 G152200),Cons2( Glyma. 17 G138500),Cons1( Glyma. 15 G270900) and Tubulin-motif( Glyma. 20 G136000) were the top four least stable genes,and they were not suitable for the study. The three housekeeping genes of Tubulin-motif( Glyma. 15 G132200),Cons6/SKIP16( Glyma. 12 G051100) and Tubulin-motif( Glyma. 05 G110200) were the most stable genes with M value of 0. 698,0. 715 and 0. 727 respectively and with their expression level of Ct = 25. 7,26. 5 and 25. 0 respectively,which indicated that they are ideal reference genes for q RT-PCR analysis under our experiment conditions.
引文
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[14]曾文韬,柴春月,窦道龙.适用于大豆实时荧光定量PCR分析的内参基因的筛选和验证[J].南京农业大学学报,2015,38(5):787-95.(Zeng W T,Zhai C Y,Dou D L.Selection and validation of reference genes for quantitative RT-PCR analysis in soybean[J].Journal of Nanjing Agricultural University,2015,38(5):787-95.)
[15]Costa J H,Saraiva K D C,Morais V D,et al.Reference gene identification for real-time PCR analyses in soybean leaves under fungus(Cercospora kikuchii)infection and treatments with salicylic and jasmonic acids[J].Australasian Plant Pathology,2016,45:191-199.
[16]Miranda V D J,Coelho R R,Viana A A B,et al.Validation of reference genes aiming accurate normalization of q PCR data in soybean upon nematode parasitism and insect attack[J].BMC Research Notes,2013,6:196.
[17]宋美静,朱晓峰,王东,等.我国大豆主产区大豆胞囊线虫群体分布及致病性分化研究[J].大豆科学,2016,35(4):630-636.(Song M J,Zhu X F,Wang D,et al.Population distribution and pathogenicity differentiation of soybean cyst nematode in main soybean production areas of china[J].Soybean Sciences,2016,35(4):630-636.)
[18]刘鹤.大豆抗胞囊线虫组织病理学及相关基因表达研究[D].沈阳:沈阳农业大学,2016:40-50(Liu H.Study on histopathology and related genes of soybean resistant to Heterodera glycines[D].Shenyang:Shenyang Agricultural University,2016:40-50.)
[19]赵思阳,王媛媛,朱晓峰,等.大豆与胞囊线虫互作中Gm PRs的表达研究[J].中国油料作物学报.2017,39(2):213-220.(Zhao S Y,Wang Y Y,Zhu X Y,et al.Expression of pathogenesis-related proteins(Gm PRs)in interactions between soybean and soybean cyst nematode[J].Chinese Journal of Oil Crop Sciences,2017,39(2):213-220.)
[20]Niblack T,Tylka G L,Arelli P,et al.A standard greenhouse method for assessing soybean cyst nematode resistance in soybean:SCE08(standardized cyst evaluation 2008)[J].Plant Health Progress,2009,10.doi:10.1094/PHP-2009-0513-01-RV.
[21]Kim H K,Kang Y J,Kim D H,et al.RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and-susceptible alleles[J].DNA Research.2011,18,483-497.
[22]Libault M,Thibivilliers S,Bilgin D D,et al.Identification of four soybean reference genes for gene expression normalization[J].The plant genome,2008,1(1):44-54.
[23]Jian B,Liu B,Bi Y,et al.Validation of internal control for gene expression study in soybean by quantitative real-time PCR[J].BMC Molecular Biology,2008,9,1-14.
[24]Sun X,Sun M,Jia B,et al.A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca2+/CAM-binding kinase Gs CBRLK and activates ROS signaling under carbonate alkaline stress[J].Plant Journal,2016,86:514-529.
[25]Stolf-Moreira R,Gertrudes de Macedo Lemos E,Vilela-Abdelnoor R,et al.Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean[J].Pesquisa Agropecuária Brasileira,2011,46:58-65.
[2]崔洪秋,冯乃杰,孙福东,等.DTA-6对大豆花荚脱落纤维素酶和Gm AC基因表达的调控[J].作物学报,2016,42(1):51-57.(Cui H Q,Feng N J,Sun F D,et al.Regulation of DTA-6by abscission cellulase and Gm AC gene expression in flowers and pods of soybean[J].ACTA Agronomica Sinica,2016,42(1):51-57.)
[3]曹鑫,邓梅,张正丽,等.小麦分蘖角度Ta TAC1基因同源克隆及表达分析.植物遗传资源学报[J],2017,18:125-132.(Cao X,Deng M,Zhang Z L,et al.Molecular characterization and expression analysis of Ta TAC1 gene in Triticum aestivum L[J].Journal of Plant Genetic Resources,2017,18:125-132.)
[4]史庆玲,董永彬,周强,等.玉米转录因子Zm ERF1的克隆及表达分析[J].生物技术进展.2017,7,38-42.(Shi Q L,Dong Y B,Zhou Q,et al.Cloning and expression analysis of Zm ERF1in maize[J].Current Biotechnology,2017,7:38-42.)
[5]王艳,武林,孙梦阳,等.不同生育时期大豆异黄酮合成相关酶基因表达的分析[J].大豆科学,2012,31:887-893.(Wang Y,Wu L,Sun M Y,et al.Analysis of gene expression underlying soybean isoflavone synthesis relative enzymes at different growth stages[J].Soybean Science,2012,31:887-893.)
[6]李媛媛,南海洋,刘宝辉,等.大豆Gm FDL06基因抗干旱及耐盐性研究[J].大豆科学,2017,36(3):351-359.(Li Y Y,Nan H Y,Liu B H,et al.Study on drought resistance and salt tolerance of soybean gene Gm FDL06[J].Soybean Science,2017,36(3):351-359.)
[7]王骁.基于实时荧光定量PCR的大豆内参基因筛选[D].太古:山西农业大学,2016:27-44.(Wang X.Selection of candidate reference genes for gene expression studies[D].Taigu:Shanxi Agricultural University,2016:27-44.)
[8]陈玉连,张涛,董登峰.低磷胁迫大豆q RT-PCR内参基因的筛选及谷胱丙肽合成代谢酶的表达[J].广西植物.2016,DOI:10.11931/guihai.gxzw201609013.(Chen Y L,Zhang T,Dong D F.Selection of reference genes for q RT-PCR and expression of genes involved in homoglutathione anabolism in soybean under stress of phosphorus deficiency[J].Gui Haia,2016,DOI:10.11931/guihai.gxzw201609013.)
[9]Ma S,Niu H,Liu C,et al.Expression stabilities of candidate reference genes for RT-q PCR under different stress conditions in soybean[J].Plos One,2013,8:e75271.
[10]刘伟灿,王骐,周永刚,等.大豆干旱胁迫下mi RNA与m RNA荧光定量PCR内参基因的筛选[J].西北农林科技大学学报(自然科学版),2016,44(2):61-67.(Liu W C,Wang Q,Zhou Y G,et al.Selection of reference genes for quantitative polymerase chain reaction of mi RNA and m RNA in soybean under drought stress[J].Journal of Northwest A&F University(Natural Science Edition),2016,44(2):61-67.)
[11]Marcolinogomes,J,Rodrigues,F A,Fugantipagliarini,R,et al.Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day[J].Plos One,2015,10(9):e0139051.
[12]Hu R,Fan C,Li H,et al.Evaluation of putative reference genes for gene expression normalization in soybean by quantitative realtime RT-PCR[J].BMC Molecular Biology,2009,10:93.
[13]侯高杰.铝毒胁迫下大豆内参基因的筛选及相关基因表达分析[D].南宁:广西大学,2015:21-32.(Hou G J.Selection of reference genes and analysis some related gene expression in soybean under aluminum toxicity[D].2015:21-32.)
[14]曾文韬,柴春月,窦道龙.适用于大豆实时荧光定量PCR分析的内参基因的筛选和验证[J].南京农业大学学报,2015,38(5):787-95.(Zeng W T,Zhai C Y,Dou D L.Selection and validation of reference genes for quantitative RT-PCR analysis in soybean[J].Journal of Nanjing Agricultural University,2015,38(5):787-95.)
[15]Costa J H,Saraiva K D C,Morais V D,et al.Reference gene identification for real-time PCR analyses in soybean leaves under fungus(Cercospora kikuchii)infection and treatments with salicylic and jasmonic acids[J].Australasian Plant Pathology,2016,45:191-199.
[16]Miranda V D J,Coelho R R,Viana A A B,et al.Validation of reference genes aiming accurate normalization of q PCR data in soybean upon nematode parasitism and insect attack[J].BMC Research Notes,2013,6:196.
[17]宋美静,朱晓峰,王东,等.我国大豆主产区大豆胞囊线虫群体分布及致病性分化研究[J].大豆科学,2016,35(4):630-636.(Song M J,Zhu X F,Wang D,et al.Population distribution and pathogenicity differentiation of soybean cyst nematode in main soybean production areas of china[J].Soybean Sciences,2016,35(4):630-636.)
[18]刘鹤.大豆抗胞囊线虫组织病理学及相关基因表达研究[D].沈阳:沈阳农业大学,2016:40-50(Liu H.Study on histopathology and related genes of soybean resistant to Heterodera glycines[D].Shenyang:Shenyang Agricultural University,2016:40-50.)
[19]赵思阳,王媛媛,朱晓峰,等.大豆与胞囊线虫互作中Gm PRs的表达研究[J].中国油料作物学报.2017,39(2):213-220.(Zhao S Y,Wang Y Y,Zhu X Y,et al.Expression of pathogenesis-related proteins(Gm PRs)in interactions between soybean and soybean cyst nematode[J].Chinese Journal of Oil Crop Sciences,2017,39(2):213-220.)
[20]Niblack T,Tylka G L,Arelli P,et al.A standard greenhouse method for assessing soybean cyst nematode resistance in soybean:SCE08(standardized cyst evaluation 2008)[J].Plant Health Progress,2009,10.doi:10.1094/PHP-2009-0513-01-RV.
[21]Kim H K,Kang Y J,Kim D H,et al.RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and-susceptible alleles[J].DNA Research.2011,18,483-497.
[22]Libault M,Thibivilliers S,Bilgin D D,et al.Identification of four soybean reference genes for gene expression normalization[J].The plant genome,2008,1(1):44-54.
[23]Jian B,Liu B,Bi Y,et al.Validation of internal control for gene expression study in soybean by quantitative real-time PCR[J].BMC Molecular Biology,2008,9,1-14.
[24]Sun X,Sun M,Jia B,et al.A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca2+/CAM-binding kinase Gs CBRLK and activates ROS signaling under carbonate alkaline stress[J].Plant Journal,2016,86:514-529.
[25]Stolf-Moreira R,Gertrudes de Macedo Lemos E,Vilela-Abdelnoor R,et al.Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean[J].Pesquisa Agropecuária Brasileira,2011,46:58-65.