利用转录组及iTRAQ技术筛选高油酸油菜抗病相关基因

doi:10.7668/hbnxb.2015.05.003

摘要/Abstract

摘要： 高油酸油菜和低油酸油菜对菌核病的抗性存在显著差异,为了弄清其分子机理,以一组高油酸油菜近等基因系自交授粉后20~35 d的种子为材料,分别进行转录组和同位素相对标记与绝对定量技术分析。分析了与抗病相关的氧化磷酸化、植物激素信号转导通路和植物病原体互作3个分类,将注释的基因和蛋白进行关联,并对其中可能与抗病相关的基因进行定量PCR验证。结合前人研究发现,基因表达或蛋白表达发生显著变化的基因gi|260505503(多聚半乳糖醛酸酶抑制蛋白)、gi|226346102(HSR203J类蛋白)、gi|470103214(钙调蛋白类)与抗病相关;而gi|297843222(结合蛋白)、gi|18397961(2-铁,2-硫-铁氧化还原类蛋白)、gi|196052306(还原型烟酰胺腺嘌呤二核苷酸脱氢酶亚基)、gi|18423437(还原型烟酰胺腺嘌呤二核苷酸脱氢酶(辅酶)1α-复形5)和gi|297794581(激酶家族蛋白)等基因差异显著。

关键词: 菌核病, 高油酸油菜, 转录组, 同位素相对标记与绝对定量技术, 定量PCR

Abstract: There are obvious differences between high oleic acid rapeseed and low oleic acid rapeseed on sclerotinia sclerotiorum resistance.In order to find out the molecule mechanism,the high oleic acid rapeseed inbred line seeds 20-35 d after pollination were used as material for transcriptome analysis and iTRAQ analysis respectively in this study.The classifications associated with disease resistance were oxidative phosphorylation,plant hormone signal transduction and plant-pathogen interaction were discussed,and the relationship between differential genes and the corresponding protein were investigated too.Then real-time quantitative PCR(qPCR)analysis was used to verify the expression levels of differentially expressed genes which may be associated with disease resistance.Combined with previous study,the genes related with disease resistance were:gi|260505503(polygalacturonase inhibitory protein),gi|226346102(HSR203J-like protein)and gi|470103214(caltractin-like).And the genes of gi|297843222(binding protein),gi|18397961(2Fe-2S ferredoxin-like protein),gi|196052306(NADH dehydrogenase subunit),gi|18423437(NADH dehydrogenase(ubiquinone)1alpha subcomplex 5)and gi|297794581(kinase family protein)have obvious difference.

Key words: Sclerotinia sclerotiorum, High oleic acid rapeseed, Transcriptome, Isobaric tags for relative and absolute quantitation(iTRAQ), Real-time quantitative PCR(qPCR)

中图分类号:

S565.4

张振乾, 肖钢, 官春云, 邬贤梦, 熊兴华, 李云昌, 胡琼, 陈社员. 利用转录组及iTRAQ技术筛选高油酸油菜抗病相关基因[J]. 华北农学报, 2015, 30(5): 16-24. doi: 10.7668/hbnxb.2015.05.003.

ZHANG Zhen-qian, XIAO Gang, GUAN Chun-yun, WU Xian-meng, XIONG Xing-hua, LI Yun-chang, HU Qiong, CHEN She-yuan. The Study of High Oleic Acid Rapeseed Disease Resistance Related Genes by Transcriptome and iTRAQ Analysis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(5): 16-24. doi: 10.7668/hbnxb.2015.05.003.

http://www.hbnxb.net/CN/Y2015/V30/I5/16

参考文献

[1] 张振乾,肖钢,谭太龙,等.高油酸油菜研究进展及其前景展望[J].作物杂志,2009(5):1-6.
[2] Jung J H,Kim H,Go Y S,et al.Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development of Brassica napus containing high oleic acid contents[J].Plant Cell Reports,2011,30(10):1881-1892.
[3] 肖钢,张宏军,彭琪,等.甘蓝型油菜油酸脱氢酶基因(fad2)多个拷贝的发现及分析[J].作物学报,2008,34(9):1563-1568.
[4] Zhang Z Q,Xiao G,Liu R Y,et al.Proteomic analysis of differentially expressed proteins between Xiangyou 15 variety and the mutant M15[J].Frontiers of Biology,2014,9(3):234-243.
[5] Suresha G S,Santha I M.Molecular cloning and in silico analysis of novel oleate desaturase gene homologues from Brassica juncea through sub-genomic library approach[J].Plant Omics,2013,6(1):55-64.
[6] Karine V,Maarten D B,Nele H,et al.Ozone effects on yield quality of spring oilseed rape and broccoli[J].Atmospheric Environment,2012,47:76-83.
[7] 官梅,李栒.高油酸油菜品系农艺性状研究[J].中国油料作物学报,2008,30(1):25-28.
[8] Liu F L,Wang W J,Sun X T,et al.RNA-Seq revealed complex response to heat stress on transcriptomic level in Saccharina japonica(Laminariales,Phaeophyta)[J].Journal of Applied Phycology,2014,26(3):1585-1596.
[9] Li Y,Wang N,Zhao F T,et al.Changes in the transcriptomic profiles of maize Roots in response to iron-deficiency stress[J].Plant Molecular Biology,2014,85(4/5):349-363.
[10] Salgado L R,Koop D M,Pinheiro D G,et al.De novo transcriptome analysis of Hevea brasiliensis tissues by RNA-seq and screening for molecular markers[J].BMC Genomics,2014,15:236.
[11] Wu W W,Wang G,Baek S J,et al.Comparative study of three protcomic quantitative methods,DIGE,cICAI and iiTRAQ,using 2D gel-or LC-MALDI TOF/TOF[J].Protcomc Res,2006,5(3):651-658.
[12] Wang J P,Mei H,Zheng C,et al.The metabolic regulation of sporulation and parasporal crystal formation in Bacillus thuringiensis revealed by transcriptomics and proteomics[J].Molecular & Cellular Proteomics,2013,12(5):1363-1376.
[13] Lan P,Li W F,Schmidt W.Complementary proteome and transcriptome profiling in phosphate-deficient Arabidopsis Roots reveals multiple levels of gene regulation[J].Molecular & Cellular Proteomics,2012,11(11):1156-1166.
[14] Dyhrman S T,Jenkins B D,Rynearson T A,et al.The transcriptome and proteome of the diatom Thalassiosira pseudonana reveal a diverse Phosphorus stress response[J].PLoS One,2012,7(3):0033768.
[15] Gonzalez L,El K W,Ju C,et al.Integrated transcriptomic and proteomic profiling of white spruce stems during thetransition from active growth to dormancy[J].Plant Cell and Environment,2012,35(4):682-701.
[16] Kuss C,Gan C S,Gunalan K,et al.Quantitative proteomics reveals new insights into erythrocyte invasion by Plasmodium falciparum[J].Molecular & Cellular Proteomics,2012,11(2):010645.
[17] Robbins M L,Roy A,Wang P H,et al.Comparative proteomics analysis by DIGE and iTRAQ provides insight into the regulation of phenylpropanoids in maize[J].Journal of Proteomics,2013,93(SI):254-275.
[18] Clark M E,He Z L,Redding A M,et al.Transcriptomic and proteomic analyses of Desulfovibrio vulgaris biofilms:Carbon and energy flow contribute to the distinct biofilm growth state[J].BMC Genomics,2012,13:138.
[19] 张秋萍.甘蓝型油菜与菌核菌互作的转录组学研究[D].长沙:湖南农业大学,2014.
[20] Wen L,Tan T L,Shu J B,et al.Using proteomic analysis to find the proteins involved in resistance against Sclerotinia sclerotiorum in adult Brassica napus[J].European Journal of Plant Pathology,2013,137(3):505-523.
[21] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2^(-ΔΔCT) Method[J].Methods(San Diego,Calif.),2001,25(4):402-408.
[22] Bauer S,Schott A K,Illarionova V,et al.Biosynthesis of tetrahydrofolate in plants:Crystal structure of 7,8-dihydroneopterin aldolase from Arabidopsis thaliana reveals a novel adolase class[J].Journal of Molecular Biology,2004,339(4):967-979.
[23] Lopez P,A Lacks S.A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities[J].J Bacteriol,1993,175(8):2214-2220.
[24] De Lorenzo G,Ferrari S.Polygalacturonase-inhibiting proteins in defense against phytopathogenic fungi[J].Current Opinion in Plant Biology,2002,5(4):295-299.
[25] Deo A,Shastri N V.Purification and characterization of polygalacturonase-inhibitory proteins from Psidium guajava Linn.(guava)fruit[J].Plant Science,2003,164(2):147-156.
[26] Azevedo C,Betsuyaku S,Peart J A,et al.Role of SGT1in resistance protein accumulation in plant immunity[J].EMBO Journal,2006,25(9):2007-2016.
[27] Tör M,Gordon P,Cuzick A,et al.Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes[J].American Society of Plant Biologists,2002,14(5):993-1003.
[28] Gomez-Gomez L,Boller T.FLS2:an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis[J].Molecular Cell,2000,5(6):1003-1011.
[29] Tang J Y,Zhu X D,Wang Y Q,et al.Semi-dominant mutations in the CC-NB-LRR-type R gene,NLS1,Lead to constitutive activation of defense responses in rice[J].Plant Journal,2011,66(6):996-1007.
[30] Forsthoefel N R,Dao T P,Vernon D M.PIRL1and PIRL9,encoding members of a novel plant-specific family of leucine-rich repeat proteins,are essential for differentiation of microspores into pollen[J].Planta,2010,232(5):1101-1114.
[31] Chico J M,Raices M,Tellez-Inon M T,et al.A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants[J].Plant Physiology,2002,128(1):256-270.
[32] 付力文.水稻钙依赖性蛋白激酶oscpk10和oscpk20在植物抗病防卫反应中的功能研究[D].北京:北京大学,2013.
[33] 赵永山.胼胝质对胞间连丝的修饰在大豆抗病毒侵染过程中的作用[D].保定:河北农业大学,2010.
[34] 姚贵滨.大豆抵抗SMV长距离运输机制的研究[D].保定:河北农业大学,2011.
[35] 吴思思,李文龙,肖东强,等.大豆不同花叶病毒抗性品种胼胝质荧光标记初探[J].植物遗传资源学报,2013,14(1):132-140.
[36] Pontier D,Godiard L,MarcoY,et al.Hsr203J,a tobacco gene whose activation is rapid,highly localized and specific for incompatible plant/pathogen interactions[J].Plant Journal,1994,5(4):507-521.
[37] Belbahri L,Boucher C,Thierry C,et al.A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant-pathogen interaction incompatible[J].The Plant Journal,2001,28(4):419-430.
[38] 周鑫.番茄叶霉菌基因 CfHNNI1 诱导植物非寄主抗病性的功能分析[D].杭州:浙江大学,2006.

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