不同抗性烟草种质对CMV侵染的细胞响应差异分析
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摘要
为解析烟草对CMV的抗病细胞学机制,以抗病品种台烟8号和感病品种NC82为材料,利用透射电子显微镜对比两品种在接种CMV后发病过程中的细胞超微结构变化。结果表明:(1)接种CMV后,台烟8号发病症状多为花叶,NC82发病症状多泡斑和深绿、褪绿区域相间;(2)对比NC82轻微花叶和泡斑的超微结构发现,二者叶绿体类囊体片层都减少,但泡斑症状细胞内线粒体嵴和CMV颗粒密度都高于轻微花叶症状细胞,说明泡斑的细胞病变程度重于花叶症状;对比NC82和台烟8号的深绿和褪绿区域细胞超微结构发现,深绿区域的叶绿体形态较完整,而褪绿区域的叶绿体严重变形、类囊体片层紊乱、淀粉粒畸形或消失,说明褪绿区域叶肉细胞病变程度重于深绿区域;(3)对比所有病变叶片中过氧化物酶、自噬小体及内膜系统结构发现,台烟8号在CMV侵染后,其三者形态结构都比NC82的更规则有序。研究结果为进一步探究抗CMV烟草品种的抗性分子机制提供理论依据。
In order to reveal the cytological mechanism of tobacco resistance to CMV, we compared the ultrastructure of the leaves between resistant variety Taiyan 8(T.T.8) and susceptible variety NC82 after CMV inoculation by transmission electronic microscopy(TEM). The results showed that(1) After CMV infection, the disease symptoms of T.T.8 were mild mosaic, while that of NC82 often were bubble spot, light green island(LGI) and dark green island(DGI).(2) Comparing the ultrastructure of mild mosaic and bubble spot in NC82, thylakoid lamellae of both symptoms were reduced, but the density of mitochondrial cristae and CMV coat particles in bubble spot cell was higher than that in slight mosaic. This indicated that the damage degree of cell structure of bubble spot was more severe than mild mosaic. Comparing the ultrastructure of LGI and DGI in NC82 and T.T.8, the structure of chloroplasts in DGI was integrated, while chloroplasts in LGI were seriously deformed, thylakoid lamellae was disordered, starch granules were abnormal or disappeared. It inferred that the degree of cell structure lesion in LGI was more serious than that in DGI.(3) Comparing to all pathological leaves, the morphology of autophagy structure, peroxisome and endomembrane system in T.T.8 were more orderly than NC82. The results from this study provide theoretical basis for further study on molecular resistance mechanisms of T.T.8.
In order to reveal the cytological mechanism of tobacco resistance to CMV, we compared the ultrastructure of the leaves between resistant variety Taiyan 8(T.T.8) and susceptible variety NC82 after CMV inoculation by transmission electronic microscopy(TEM). The results showed that(1) After CMV infection, the disease symptoms of T.T.8 were mild mosaic, while that of NC82 often were bubble spot, light green island(LGI) and dark green island(DGI).(2) Comparing the ultrastructure of mild mosaic and bubble spot in NC82, thylakoid lamellae of both symptoms were reduced, but the density of mitochondrial cristae and CMV coat particles in bubble spot cell was higher than that in slight mosaic. This indicated that the damage degree of cell structure of bubble spot was more severe than mild mosaic. Comparing the ultrastructure of LGI and DGI in NC82 and T.T.8, the structure of chloroplasts in DGI was integrated, while chloroplasts in LGI were seriously deformed, thylakoid lamellae was disordered, starch granules were abnormal or disappeared. It inferred that the degree of cell structure lesion in LGI was more serious than that in DGI.(3) Comparing to all pathological leaves, the morphology of autophagy structure, peroxisome and endomembrane system in T.T.8 were more orderly than NC82. The results from this study provide theoretical basis for further study on molecular resistance mechanisms of T.T.8.
引文
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[15]文柳璎,刘旦,龚敏,等.抗病毒病K326新品系Y48抗TMV的细胞学机制研究[J].中国烟草科学,2018,39(4):18-25.WEN L Y,LIU D,GONG M,et al.Analysis of cytological mechanism of TMV resistance in novel anti-viral disease variety developed from K326[J].Chinese Tobacco Science,2018,39(4):18-25.
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[17]QUEIROS F,RODRIGUES J A,ALMEIDA J M,et al.Differential responses of the antioxidant defense system and ultrastructure in a salt-adapted potato cell line[J].Plant Physiology and Biochemistry,2011,49(12):1410-1419.
[18]SUN L J,REN H Y,LIU R X,et al.An h-type thioredoxin functions in tobacco defense responses to two species of viruses and an abiotic oxidative stress[J].Mol Plant-Microbe Interact,2010,23(11):1470-1485.
[19]TADA Y,SPOEL S H,PAJEROWSKA-MUKHTAR K,et al.Plant immunity requires conformational charges of NPR1 via S-nitrosylation and thioredoxins[J].Science,2008,321(5891):952-956.
[20]SCHAAD M C,JENSEN P E,CARRINGTON J C.Formation of plant RNA virus replication complexes on membranes:role of an endoplasmic reticulum-targeted viral protein[J].EMBO Journal,2014,16:4049-4059.
[21]MAGLIANO D,MARSHALL J A,BOWDEN D S,et al.Rubella virus replication complexes are virus-modified lysosomes[J].Virology,1998,240:57-63
[22]LEE W M,ISHIKAWA M,AHLQUIST P.Mutation of hostδ9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNAsynthesis[J].Journal of Virology,2001,75(5):2097-2106
[23]JHENG J R,HO J Y,HORNG J T.ER stress,autophagy,and RNA viruses[J].Frontiers in Microbiology,2014,5:388.
[24]刘伟,李方方,孙航军,等.TMV侵染烟草诱导寄主产生细胞自噬[J].植物病理学报,2016,46(6):759-766.LIU W,LI F F,SUN H J,et al.Tobacco mosaic virus infection on tobacco plants induces autophagy[J].Acta Phytopathologica Sinica,2016,46(6):759-766.
[25]LI L,WANG L,XIAO R,et al.The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells[J].Bioscience Reports,2012,32(Pt 2):171.
[26]任民,程立锐,刘旦,等.基于RAD重测序技术开发烟草品种SNP位点[J].中国烟草科学,2018,39(3):10-17.REN M,CHENG L R,LIU D,et al.Development of tobacco SNPs based on RAD re-sequencing technology[J].Chinese Tobacco Science,2018,39(3):10-17.
[27]潘旭浩,程立锐,陈小翠,等.基于SLAF-seq技术的烟草抗CMV主效QTL定位[J].中国烟草科学,2018,39(5):4-11.PAN X H,CHENG L R,CHEN X C,et al.CMVresistance QTL analysis in tobacco based on SALF-seq technique[J].Chinese Tobacco Science,2018,39(5):4-11.
[2]范静苑,王元英,蒋彩虹,等.烟草CMV抗性鉴定及抗性基因的SSR标记研究[J].分子植物育种,2009,7(2):355-359.FAN J Y,WANG Y Y,JIANG C H,et al.Resistance identification and SSR marking of resistance gene of tobacco cucumber mosaic virus[J].Molecular Plant Breeding,2009,7(2):355-359.
[3]胡向武,陈士超,张林普.黄瓜花叶病毒的侵染扩散及病变细胞的电镜观察[J].安徽师大学报(自然科学版),1998,21(4):354-357.HU X W,CHEN S C,ZHANG L P.Observation on the invasion and diffusion of cmv particles and pathologic changes of cells under the electron microscope[J].Journal of Anhui Normal University,1998,21(4):354-357.
[4]CHEN L J,LIU J,ZHAO F F,et al.Characterisation of the dark green islands of cucumber mosaic virus infected Nicotianatabacum[J].Plant Cell Report,2015,34(7):1225-1238.
[5]申莉莉.CMV诱导烟草内质网应激及调控因子Nbb ZIP28的研究[D].沈阳:沈阳农业大学,2017.SHEN L L.Endoplasmic reticulum stress induced by CMV and research on the regulator NbbZIP28 in Nicotiana[D].Shenyang:Shenyang Agricultural University,2017.
[6]李方方,申莉莉.植物病毒侵染诱导寄主内质网应激反应[J].中国烟草科学,2016,37(6):95-100.LI F F,SHEN L L,Endoplasmic reticulum stress induced by plant viral infection[J].Chinese Tobacco Science,2016,37(6):95-100.
[7]HAASE A,RICHTER J,RABENSTEIN F.Monoclonal antibodies for detection and serotyping of cucumber mosaic virus[J].Journal of Phytopathology,2010,127(2):129-136.
[8]MOCHIZUKI T,OHKI S T.Single amino acid substitutions at residue 129 in the coat protein of cucumber mosaic virus affect symptom expression and thylakoid structure[J].Archives of virology,2011,156:881-886.
[9]TAKAHASHI H,SUZUKI M,NATSUAKI K,et al.Mapping the virus and host genes involved in the resistance response in Cucumber mosaic virus-infected Arabidopsis thaliana[J].Plant cell physiology,2001,42:340-347.
[10]金大伟.我国主要烟区烟草黄瓜花叶病毒的鉴定及全基因组序列分析[D].武汉:华中农业大学,2014.JIN D W.Identification and complete sequence analysis of cucumber mosaic virus from tobacco in the main tobacco growing areas in china[D].Wuhan:Huazhong Agricultural University,2014.
[11]宋丽云.我国烟草TMV和CMV种群结构遗传分析[D].北京:中国农业科学院,2012.SONG L Y.Phylogeographic analysis of TMV and CMVof Tobacco in china[D].Beijing:Chinese Academy of Agricultural Sciences,2012.
[12]陈小翠,代帅帅,张兴伟,等.烤烟CMV抗性的主基因+多基因混合遗传模型分析[J].植物遗传资源学报,2014,15(6):1278-1286.CHEN X C,DAI S S,ZHANG X W,et al.Mixed major-gene plus polygenes inheritance analysis for CMVdisease resistance in flue-cured tobacco[J].Journal of Plant Genetic Resources,2014,15(6):1278-1286.
[13]程立锐,陈小翠,代帅帅,等.烟草黄瓜花叶病毒抗性位点发掘[J].中国烟草科学,2018,39(4):1-6.CHENG L R,CHEN X C,DAI S S,et al.Identification of QTL associated with resistance to cucumber mosaic virus in tobacco[J].Chinese Tobacco Science,2018,39(4):1-6.
[14]徐鹏飞,吴俊江,XUE A G,等.大豆疫霉根腐病菌游动孢子侵染野生大豆下胚轴的透射电镜观察[J].大豆科学,2012,31(3):462-465.XU P F,WU J J,XUE A G,et al.Ultrastructure of hypocotyls of glycine soja infected with zoospores of phytophthorasojae[J].Soybean Science,2012.31(3):462-465.
[15]文柳璎,刘旦,龚敏,等.抗病毒病K326新品系Y48抗TMV的细胞学机制研究[J].中国烟草科学,2018,39(4):18-25.WEN L Y,LIU D,GONG M,et al.Analysis of cytological mechanism of TMV resistance in novel anti-viral disease variety developed from K326[J].Chinese Tobacco Science,2018,39(4):18-25.
[16]TRIPATHI D N,WALKER C L.The peroxisome as a cell signaling organelle[J].Current Opinion in Cell Biology,2016,39:109-112.
[17]QUEIROS F,RODRIGUES J A,ALMEIDA J M,et al.Differential responses of the antioxidant defense system and ultrastructure in a salt-adapted potato cell line[J].Plant Physiology and Biochemistry,2011,49(12):1410-1419.
[18]SUN L J,REN H Y,LIU R X,et al.An h-type thioredoxin functions in tobacco defense responses to two species of viruses and an abiotic oxidative stress[J].Mol Plant-Microbe Interact,2010,23(11):1470-1485.
[19]TADA Y,SPOEL S H,PAJEROWSKA-MUKHTAR K,et al.Plant immunity requires conformational charges of NPR1 via S-nitrosylation and thioredoxins[J].Science,2008,321(5891):952-956.
[20]SCHAAD M C,JENSEN P E,CARRINGTON J C.Formation of plant RNA virus replication complexes on membranes:role of an endoplasmic reticulum-targeted viral protein[J].EMBO Journal,2014,16:4049-4059.
[21]MAGLIANO D,MARSHALL J A,BOWDEN D S,et al.Rubella virus replication complexes are virus-modified lysosomes[J].Virology,1998,240:57-63
[22]LEE W M,ISHIKAWA M,AHLQUIST P.Mutation of hostδ9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNAsynthesis[J].Journal of Virology,2001,75(5):2097-2106
[23]JHENG J R,HO J Y,HORNG J T.ER stress,autophagy,and RNA viruses[J].Frontiers in Microbiology,2014,5:388.
[24]刘伟,李方方,孙航军,等.TMV侵染烟草诱导寄主产生细胞自噬[J].植物病理学报,2016,46(6):759-766.LIU W,LI F F,SUN H J,et al.Tobacco mosaic virus infection on tobacco plants induces autophagy[J].Acta Phytopathologica Sinica,2016,46(6):759-766.
[25]LI L,WANG L,XIAO R,et al.The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells[J].Bioscience Reports,2012,32(Pt 2):171.
[26]任民,程立锐,刘旦,等.基于RAD重测序技术开发烟草品种SNP位点[J].中国烟草科学,2018,39(3):10-17.REN M,CHENG L R,LIU D,et al.Development of tobacco SNPs based on RAD re-sequencing technology[J].Chinese Tobacco Science,2018,39(3):10-17.
[27]潘旭浩,程立锐,陈小翠,等.基于SLAF-seq技术的烟草抗CMV主效QTL定位[J].中国烟草科学,2018,39(5):4-11.PAN X H,CHENG L R,CHEN X C,et al.CMVresistance QTL analysis in tobacco based on SALF-seq technique[J].Chinese Tobacco Science,2018,39(5):4-11.