2个抗病相关基因与高油酸油菜病害关系分析
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摘要
为快速筛选抗病性强的高油酸油菜品种,本研究以20个高油酸油菜品系为试验材料,调查不同材料的抗病情况,并通过RT-qPCR测定经miRNA测序筛选得到的与抗病相关基因HSP90和ATG3的表达情况,同时分析其与病情指数的关系。结果表明,20个高油酸油菜品系的发病率与病情指数呈线性正相关关系,且差异极显著; HSP90基因从苗期至花期表达量呈逐渐降低的趋势,角果期有先升高后降低和逐渐升高2种表达模式; ATG3基因从苗期至花期表达量也呈逐渐升高的趋势,角果期表达模式与HSP90基因相同。用HSP90基因在花期-叶中表达量≥1. 5倍内参时进行抗病材料筛选,有95%的准确率;用ATG3基因在花期-叶中表达量≥1. 8倍内参时进行抗病材料筛选,有80%的准确率,此外,也可用HSP90在花期-叶结合ATG3在五～六叶期-叶中表达情况进行预测,有80%的准确率。本研究结果为高油酸油菜抗病材料的早期筛选和油酸油菜育种提供了一定的理论依据。
In order to rapid screen strongly resistant high oleic acid rapeseed varieties,In this study,20 high oleic acid rapeseed varieties were used as materials,the disease resistance of different materials was investigated,and diseaseresistance related genes expression of HSP90 and ATG3 which screened from microRNA sequencing and detected by RTq PCR,then the relationship between related genes and disease index were analyzed.The results showed that there was a significantly linear positive correlation between incidence and disease index of 20 high oleic acid rapeseed varieties,the expression of HSP90 decreased gradually until the flowering stage,and there were two kinds of expression patterns in the pod stage with Low-high-low and gradually decreased. ATG3 gene expression increased gradually until the flowering stage,and also appeared the same expression patterns with HSP90 in pod stage. When the expression of HSP90 was more than 1. 5 times of the reference gene in florescence-leaf,the disease-resistant materials were screened with 95%accuracy. Similarly,when the expression of ATG3 was more than 1. 8 times of the reference gene in florescence-flowers,the disease-resistant materials were screened with 80% accuracy. In addition,to combine the expression of HSP90 in florescence-leaf and ATG3 in 5-6 leaf stage leaves can also be used to predict the disease-resistant materials( 80%accuracy). The results of this study provids a certain theoretical foundation for screening high oleic acid rapeseed materials with strong disease-resistance in the early stages and promoting the breeding of high oleic acid rapeseed.
In order to rapid screen strongly resistant high oleic acid rapeseed varieties,In this study,20 high oleic acid rapeseed varieties were used as materials,the disease resistance of different materials was investigated,and diseaseresistance related genes expression of HSP90 and ATG3 which screened from microRNA sequencing and detected by RTq PCR,then the relationship between related genes and disease index were analyzed.The results showed that there was a significantly linear positive correlation between incidence and disease index of 20 high oleic acid rapeseed varieties,the expression of HSP90 decreased gradually until the flowering stage,and there were two kinds of expression patterns in the pod stage with Low-high-low and gradually decreased. ATG3 gene expression increased gradually until the flowering stage,and also appeared the same expression patterns with HSP90 in pod stage. When the expression of HSP90 was more than 1. 5 times of the reference gene in florescence-leaf,the disease-resistant materials were screened with 95%accuracy. Similarly,when the expression of ATG3 was more than 1. 8 times of the reference gene in florescence-flowers,the disease-resistant materials were screened with 80% accuracy. In addition,to combine the expression of HSP90 in florescence-leaf and ATG3 in 5-6 leaf stage leaves can also be used to predict the disease-resistant materials( 80%accuracy). The results of this study provids a certain theoretical foundation for screening high oleic acid rapeseed materials with strong disease-resistance in the early stages and promoting the breeding of high oleic acid rapeseed.
引文
[1]李丽丽.世界油菜病害研究概述[J].中国油料作物学报,1994(1):79-81
[2]Uloth M B,Clode P L,You M P,Barbetti M J.Attack modes and defence reactions in pathosystems involving Sclerotinia sclerotiorum,Brassica carinata,B.juncea and B.napus[J].Annals of Botany,2016,117(1):79-95
[3]Lydiate D J,Pilcher R L,Higgins E E,Walsh J A.Genetic control of immunity to Turnip mosaic virus(Tu MV)pathotype 1 in Brassica rapa(Chinese cabbage)[J].Genome,2014,57(8):419
[4]Carlsson M,Von B R,Merker A.Screening and evaluation of resistance to downy mildew(Peronospora parasitica)and clubroot(Plasmodiophora brassicae)in genetic resources of Brassica oleracea[J].Hereditas,2010,141(3):293-300
[5]官春云.优质油菜生理生态和现代栽培技术[M].北京:中国农业出版社,2013
[6]Guan C Y,Liu C L,Chen S Y,Peng Q,Li X,Guan M.High oleic acid content materials of rapeseed(Brassica napus)produced by radiation breeding[J].Acta Agronomica Sinica,2006,32(11):1625-1629
[7]张振乾,胡庆一,官春云.高油酸油菜研究现状、存在的问题及发展建议[J].作物研究,2016,30(4):462-474
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[9]Jin H J,Kim H,Go Y S,Lee S B,Hur C G,Kim H U,Suh M C.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
[10]郎春秀,王伏林,吴学龙,石江华,陈锦清,吴关庭.油菜种子低亚油酸和亚麻酸含量异交稳定技术的建立[J].核农学报,2016,30(9):1716-1721
[11]Wells R,Trick M,Soumpourou E,Clissold L,Morgan C,Werner P,Gibbard C,Clarke M,Jennaway R,BancroftⅠ.The control of seed oil polyunsaturate content in the polyploid crop species Brassica napus[J].Molecular Breeding,2014,33(2):349-362
[12]Niu J S,Liu J,Ma W B,Li Q Y,Wang Z Y,He D X.The relationship of methyl jasmonate enhanced powdery mildew resistance in wheat and the expressions of 9 disease resistance related genes[J].Agricultural Science&Technology,2011,12(4):504-508
[13]黄启秀,曲延英,姚正培,李梦雨,陈全家.海岛棉枯萎病抗性与类黄酮代谢途径基因表达量的相关性[J].作物学报,2017,43(12):1791-1801
[14]谷晓娜,刘振库,王丕武,张卓,马建,曲静,张君,付永平,卢实,郑成忠,刘婷婷.hrpZPsta转基因大豆中定量表达与疫霉根腐病和灰斑病抗性相关研究[J].中国油料作物学报,2015,37(1):35-40
[15]徐明月,肖庆生,张学昆,程勇,陆光远,徐劲松,曾柳,付桂萍,翟桢,邹锡玲.油菜干旱相关基因的表达及其与耐旱生理指标的相关性[J].中国油料作物学报,2013,35(5):557
[16]邓婧.甘蓝型油菜fad2基因差异表达与脂肪酸合成的相关性分析[D].长沙:湖南农业大学,2008
[17]裴丽丽,徐兆师,尹丽娟,李连城,陈明,郭玉华,马有志.植物热激蛋白90的分子作用机理及其利用研究进展[J].植物遗传资源学报,2013,14(1):109-114
[18]Dong X,Yi H,Lee J,NouⅠS,Han C T,Hur Y.Global GeneExpression Analysis to Identify Differentially Expressed Genes Critical for the Heat Stress Response in Brassica rapa[J].PLoSOne,2015,10(6):e0130451
[19]Prodromou C.Mechanisms of Hsp90 regulation[J].Biochemical Journal,2016,473(16):2439-2452
[20]Wang P,Sun X,Jia X,Ma F.Apple autophagy-related protein MdATG3s afford tolerance to multiple abiotic stresses[J].Plant Science,2017,256:53-64
[21]刘洋,张静,王秋玲,侯岁稳.植物细胞自噬研究进展[J].植物学报,2018,53(1):5-16
[22]Rose T L,Bonneau L,Der C,Martymazars D,Marty F.Starvationinduced expression of autophagy-related genes in Arabidopsis[J].Biology of the Cell,2012,98(1):53-67
[23]刘胜毅.油菜抗菌核病草酸鉴定方法[J].中国油料,1994(增刊):75-76
[24]刘芳,刘睿洋,彭烨,官春云.甘蓝型油菜BnFAD2-C1基因全长序列的克隆、表达及转录调控元件分析[J].作物学报,2015,41(11):1663-1670
[25]王晓丹,胡庆一,张振乾,王悦,官春云.不同肥密条件对甘蓝型油菜叶绿素含量及脂肪酸合成相关基因表达量影响的研究[J].华北农学报,2018,33(1):127-134
[26]Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):45
[27]秦虎强,高小宁,韩青梅,黄丽丽,张吉昌,唐建祥.油菜菌核病发生流行与菌源量、气候因子关系分析及病情预测模型的建立[J].植物保护学报,2018,45(3):496-502
[28]de Winter J C,Gosling S D,Potter J.Comparing the Pearson and Spearman correlation coefficients across distributions and sample sizes:A tutorial using simulations and empirical data[J].Psychol Methods,2016,21(3):273-90
[29]王亚军.相关系数与决定系数辨析[C]//中国科学技术期刊编辑学会.长江流域暨西北地区科技期刊协作网2008年学术年会论文集.西安:科技编辑研究,2008:74-77
[30]种康,谭克辉,白书农.高等植物开花调控的分子基础[M].北京:科学出版社,1999:563-578
[31]马田田.甘蓝型油菜抗菌核病QTL定位及相关基因表达分析[D].南京:南京农业大学,2012
[32]李冬艳.番茄抗多种病害及耐贮种质资源的筛选[D].哈尔滨:东北农业大学,2016
[33]Barkley N A,Chamberlin K D C,Wang M L,Pittman R N.Development of a real-time PCR genotyping assay to identify high oleic acid peanuts(Arachis hypogaea L.)[J].Molecular Breeding,2010,25(3):541-548
[34]Barkley N A,Wang M L,Pittman R N.A real-time PCRgenotyping assay to detect FAD2A SNPs in peanuts(Arachis hypogaea L.)[J].Electronic Journal of Biotechnology,2011,14(1):9-10
[35]杜建中,孙毅,王景雪,郝曜山,雷海英.转基因抗矮花叶病玉米的遗传、表达及抗病性研究[J].生物技术通讯,2008,19(1):43-46
[2]Uloth M B,Clode P L,You M P,Barbetti M J.Attack modes and defence reactions in pathosystems involving Sclerotinia sclerotiorum,Brassica carinata,B.juncea and B.napus[J].Annals of Botany,2016,117(1):79-95
[3]Lydiate D J,Pilcher R L,Higgins E E,Walsh J A.Genetic control of immunity to Turnip mosaic virus(Tu MV)pathotype 1 in Brassica rapa(Chinese cabbage)[J].Genome,2014,57(8):419
[4]Carlsson M,Von B R,Merker A.Screening and evaluation of resistance to downy mildew(Peronospora parasitica)and clubroot(Plasmodiophora brassicae)in genetic resources of Brassica oleracea[J].Hereditas,2010,141(3):293-300
[5]官春云.优质油菜生理生态和现代栽培技术[M].北京:中国农业出版社,2013
[6]Guan C Y,Liu C L,Chen S Y,Peng Q,Li X,Guan M.High oleic acid content materials of rapeseed(Brassica napus)produced by radiation breeding[J].Acta Agronomica Sinica,2006,32(11):1625-1629
[7]张振乾,胡庆一,官春云.高油酸油菜研究现状、存在的问题及发展建议[J].作物研究,2016,30(4):462-474
[8]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
[9]Jin H J,Kim H,Go Y S,Lee S B,Hur C G,Kim H U,Suh M C.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
[10]郎春秀,王伏林,吴学龙,石江华,陈锦清,吴关庭.油菜种子低亚油酸和亚麻酸含量异交稳定技术的建立[J].核农学报,2016,30(9):1716-1721
[11]Wells R,Trick M,Soumpourou E,Clissold L,Morgan C,Werner P,Gibbard C,Clarke M,Jennaway R,BancroftⅠ.The control of seed oil polyunsaturate content in the polyploid crop species Brassica napus[J].Molecular Breeding,2014,33(2):349-362
[12]Niu J S,Liu J,Ma W B,Li Q Y,Wang Z Y,He D X.The relationship of methyl jasmonate enhanced powdery mildew resistance in wheat and the expressions of 9 disease resistance related genes[J].Agricultural Science&Technology,2011,12(4):504-508
[13]黄启秀,曲延英,姚正培,李梦雨,陈全家.海岛棉枯萎病抗性与类黄酮代谢途径基因表达量的相关性[J].作物学报,2017,43(12):1791-1801
[14]谷晓娜,刘振库,王丕武,张卓,马建,曲静,张君,付永平,卢实,郑成忠,刘婷婷.hrpZPsta转基因大豆中定量表达与疫霉根腐病和灰斑病抗性相关研究[J].中国油料作物学报,2015,37(1):35-40
[15]徐明月,肖庆生,张学昆,程勇,陆光远,徐劲松,曾柳,付桂萍,翟桢,邹锡玲.油菜干旱相关基因的表达及其与耐旱生理指标的相关性[J].中国油料作物学报,2013,35(5):557
[16]邓婧.甘蓝型油菜fad2基因差异表达与脂肪酸合成的相关性分析[D].长沙:湖南农业大学,2008
[17]裴丽丽,徐兆师,尹丽娟,李连城,陈明,郭玉华,马有志.植物热激蛋白90的分子作用机理及其利用研究进展[J].植物遗传资源学报,2013,14(1):109-114
[18]Dong X,Yi H,Lee J,NouⅠS,Han C T,Hur Y.Global GeneExpression Analysis to Identify Differentially Expressed Genes Critical for the Heat Stress Response in Brassica rapa[J].PLoSOne,2015,10(6):e0130451
[19]Prodromou C.Mechanisms of Hsp90 regulation[J].Biochemical Journal,2016,473(16):2439-2452
[20]Wang P,Sun X,Jia X,Ma F.Apple autophagy-related protein MdATG3s afford tolerance to multiple abiotic stresses[J].Plant Science,2017,256:53-64
[21]刘洋,张静,王秋玲,侯岁稳.植物细胞自噬研究进展[J].植物学报,2018,53(1):5-16
[22]Rose T L,Bonneau L,Der C,Martymazars D,Marty F.Starvationinduced expression of autophagy-related genes in Arabidopsis[J].Biology of the Cell,2012,98(1):53-67
[23]刘胜毅.油菜抗菌核病草酸鉴定方法[J].中国油料,1994(增刊):75-76
[24]刘芳,刘睿洋,彭烨,官春云.甘蓝型油菜BnFAD2-C1基因全长序列的克隆、表达及转录调控元件分析[J].作物学报,2015,41(11):1663-1670
[25]王晓丹,胡庆一,张振乾,王悦,官春云.不同肥密条件对甘蓝型油菜叶绿素含量及脂肪酸合成相关基因表达量影响的研究[J].华北农学报,2018,33(1):127-134
[26]Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research,2001,29(9):45
[27]秦虎强,高小宁,韩青梅,黄丽丽,张吉昌,唐建祥.油菜菌核病发生流行与菌源量、气候因子关系分析及病情预测模型的建立[J].植物保护学报,2018,45(3):496-502
[28]de Winter J C,Gosling S D,Potter J.Comparing the Pearson and Spearman correlation coefficients across distributions and sample sizes:A tutorial using simulations and empirical data[J].Psychol Methods,2016,21(3):273-90
[29]王亚军.相关系数与决定系数辨析[C]//中国科学技术期刊编辑学会.长江流域暨西北地区科技期刊协作网2008年学术年会论文集.西安:科技编辑研究,2008:74-77
[30]种康,谭克辉,白书农.高等植物开花调控的分子基础[M].北京:科学出版社,1999:563-578
[31]马田田.甘蓝型油菜抗菌核病QTL定位及相关基因表达分析[D].南京:南京农业大学,2012
[32]李冬艳.番茄抗多种病害及耐贮种质资源的筛选[D].哈尔滨:东北农业大学,2016
[33]Barkley N A,Chamberlin K D C,Wang M L,Pittman R N.Development of a real-time PCR genotyping assay to identify high oleic acid peanuts(Arachis hypogaea L.)[J].Molecular Breeding,2010,25(3):541-548
[34]Barkley N A,Wang M L,Pittman R N.A real-time PCRgenotyping assay to detect FAD2A SNPs in peanuts(Arachis hypogaea L.)[J].Electronic Journal of Biotechnology,2011,14(1):9-10
[35]杜建中,孙毅,王景雪,郝曜山,雷海英.转基因抗矮花叶病玉米的遗传、表达及抗病性研究[J].生物技术通讯,2008,19(1):43-46