细胞自噬相关基因在水稻纹枯病菌菌核发育过程中的定量分析
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摘要
在前期研究中,采用RNA-seq技术获得了在水稻纹枯病菌菌核发育过程中差异表达的6个自噬相关基因,为阐明这些基因在水稻纹枯病菌菌核发育过程中的表达模式,提取了菌核发育过程中3个阶段的RNA,根据特异基因的序列设计引物,采用实时荧光定量PCR技术分析这些基因的表达谱。结果表明,丝氨酸/苏氨酸激酶和ssp1、ATG13和Vam6基因的表达量在白色菌核形成阶段的表达量显著升高,而在菌核发育成熟阶段的表达量明显下降。而丝氨酸/苏氨酸-蛋白磷酸酶基因和丝氨酸/苏氨酸激酶受体相关蛋白基因的表达量在菌核发育阶段呈现出连续下降的趋势。研究发现这6个与细胞自噬有关的基因的表达谱与转录组测序的结果一致,表明转录组测序的结果可靠;同时也表明了这些基因高度响应了菌核的发育过程。
In a previous study,six autophagy-related genes were identified during sclerotial development of Rhizoctonia solani AG1-IA using RNA-seq technology.In order to reveal the expression patterns of these genes during sclerotial development of R.solani AG1-IA,quantitative real-time PCR(qRTPCR)was performed.The results showed that expression of the serine/threonine kinase,ssp1,ATG13 and Vam6 genes was significantly up-regulated at the sclerotial initiation stage,but down-regulated at the sclerotial maturation stage.However,the serine/threonine-protein phosphatase gene and serine/threonine kinase receptor-related protein gene showed a significant decrease during sclerotial development of R.solani AGI-IA.The expression patterns of these genes obtained by qRT-PCR were in accordance with the results obtained by RNA-seq,which indicated that the results of RNA-seq were reliable.This study also showed that autophagy-related genes were highly regulated during sclerotial development,and may play an important role in sclerotial development.
In a previous study,six autophagy-related genes were identified during sclerotial development of Rhizoctonia solani AG1-IA using RNA-seq technology.In order to reveal the expression patterns of these genes during sclerotial development of R.solani AG1-IA,quantitative real-time PCR(qRTPCR)was performed.The results showed that expression of the serine/threonine kinase,ssp1,ATG13 and Vam6 genes was significantly up-regulated at the sclerotial initiation stage,but down-regulated at the sclerotial maturation stage.However,the serine/threonine-protein phosphatase gene and serine/threonine kinase receptor-related protein gene showed a significant decrease during sclerotial development of R.solani AGI-IA.The expression patterns of these genes obtained by qRT-PCR were in accordance with the results obtained by RNA-seq,which indicated that the results of RNA-seq were reliable.This study also showed that autophagy-related genes were highly regulated during sclerotial development,and may play an important role in sclerotial development.
引文
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[2]郭明亮.中国水稻氮过量对农药用量的影响[D].北京:中国农业大学,2016.
[3]王伟,杨惠,王政逸,等.在水稻纹枯病菌菌核形成中5个基因的表达差异分析[J].浙江大学学报(农业与生命科学版),2013,39(1):18-25.
[4]王玲,左示敏,张亚芳,等.中国南方八省(自治区)水稻纹枯病菌群体遗传结构的SSR分析[J].中国农业科学,2015,48(13):2538-2548.
[5]胡春锦,魏源文,黄思良,等.与广西水稻纹枯病相关菌系的鉴定和遗传特性研究(英文)[J].西北农业学报,2010,19(2):45-51.
[6]曹琦琦.立枯丝核菌不同融合群G蛋白β亚基基因克隆与功能分析[D].广州:华南农业大学,2012.
[7]FENG S,SHU C,WANG C,et al.Survival of Rhizoctonia solani AG-1IA,the causal agent of rice sheath blight,under different environmental conditions[J].Journal of phytopathology,2017,165:44-52.
[8]陈科,程汉华,周荣家.自噬与泛素化蛋白降解途径的分子机制及其功能[J].遗传,2012,34(1):7-20.
[9]李文,魏科,冯杜.自噬体膜的来源[J].中国生物化学与分子生物学报,2014,30(10):957-962.
[10]闫思源,姜学军.细胞自噬及真菌中自噬研究概述[J].菌物学报,2015,34(5):871-879.
[11]陈永.酿酒酵母中Rab蛋白Vps21及其上下游蛋白在细胞自噬中的功能研究[D].南京:南京农业大学,2014.
[12]王凯,郑洋,邢雅玲,等.细胞自噬在病毒感染与免疫调节中的作用机制[J].中国生物化学与分子生物学报,2011,27(11):987-992.
[13]MATSUURA A,TSUKADA M,WADA Y,et al.Apg1p,a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae[J].Gene,1997,192(2):245-250.
[14]李超萍,林春花,翟李刚,等.橡胶树胶孢炭疽病菌致病相关基因CgATG8的功能分析[J].热带作物学报,2013,34(11):2172-2178.
[15]RICHIE D L,FULLER K K,FORTWENDEL J,et al.Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus[J].Eukaryotic cell,2007,6(12):2437-2447.
[16]LIU T B,LIU X H,LU J P,et al.The cysteine protease MoAtg4interacts with MoAtg8and is required for differentiation and pathogenesis in Magnaporthe oryzae[J].Autophag,2010,6(1):74-85.
[17]曾晓清.稻瘟病菌SNF1途径和细胞自噬蛋白MoAtg14的功能分析[D].杭州:浙江大学,2014.
[18]舒灿伟,陈健仪,赵美,等.一种优化的适用于双向电泳的水稻纹枯病菌蛋白质提取方法[J].华中农业大学学报,2017,36(5):15-19.
[19]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[20]马亚男.Rhizoctonia spp.菌核分化不同阶段活性氧代谢及相关酶变化[D].泰安:山东农业大学,2011.
[21]GEORGIOU C D,PATSOUKIS N,PAPAPOSTOLOU I,et al.Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress[J].Integrative&comparative biology,2006,46(6):691-712.
[22]王玉林.禾谷镰刀菌丝/苏氨酸蛋白激酶SCH9基因的功能研究[D].杨凌:西北农林科技大学,2011.
[23]彭斌,王静,胡源,等.丝氨酸/苏氨酸蛋白磷酸酶与DNA损伤应答[J].生命科学,2014(11):1120-1135.
[24]王林纤,戴勇,彭武建,等.丝氨酸-苏氨酸激酶受体相关蛋白在健康人和系统性红斑狼疮患者外周血单个核细胞中的差异表达[J].第三军医大学学报,2011,33(2):148-151.
[25]刘蒙蒙,邢咏梅,郭顺星.猪苓菌核自噬相关基因ATG8的克隆及表达分析[J].中国医药生物技术,2016,11(5):415-419.
[26]高惠敏.稻瘟病菌液泡运输途径相关基因MoYpt7和MoMon1的克隆和功能分析[D].杭州:浙江大学,2012.