核盘菌γ-谷氨酰磷酸还原酶基因SsGPR1的功能分析
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摘要
【目的】γ-谷氨酰磷酸还原酶是真菌脯氨酸合成途径中的一个关键性酶,本研究旨在对核盘菌(Sclerotinia sclerotiorum)γ-谷氨酰磷酸还原酶编码基因SsGPR1进行沉默,并对沉默转化子菌丝生长、菌核形成和致病力等表型进行研究,为揭示核盘菌的生长发育与致病机理打下基础,并为作物菌核病的绿色防控提供线索。【方法】通过BLAST进行蛋白同源比对分析,并利用MEGA 5.0软件构建蛋白进化树。通过实时荧光RT-PCR检测SsGPR1在核盘菌菌丝生长、菌核发育和萌发的各个阶段以及致病不同时期的表达模式。根据RNA干扰原理构建SsGPR1的沉默载体,通过PEG介导原生质体转化的方法将沉默载体转入到野生型核盘菌菌株1980中。利用实时荧光RT-PCR鉴定基因沉默转化子,对沉默转化子的菌丝形态、生长速度和菌核形成等表型进行观察,并测定沉默转化子在氧化胁迫条件下的菌丝生长。将沉默转化子分别接种至活体油菜叶片和拟南芥植株,观察并测量病斑大小。通过酸性茚三酮法对沉默转化子中的游离脯氨酸进行测定。【结果】核盘菌SsGPR1全长1 454 bp,编码449个氨基酸,在氨基酸H~(10)-N~(426)处含有谷氨酰磷酸还原酶结构域。同源比对发现SsGPR1蛋白与灰葡萄孢(Botrytis cinerea)的BC1G_13183蛋白和白霉病菌(Sclerotinia borealis)的SBOR_2215蛋白相似性最高,氨基酸序列一致性分别达95%和94%,系统进化树结果表明三者聚为一个小的分支。SsGPR1在核盘菌菌丝生长时期的表达量较高,在菌核不同发育阶段表达量相近,但均低于菌丝生长时期。SsGPR1在致病时期表达量不断升高,在接种后9 h表达量最高。将SsGPR1基因沉默载体pSIGPR1导入到核盘菌野生型菌株中,并通过实时荧光RT-PCR检测不同转化子中SsGPR1的表达水平,结果表明SiGPR1-104和SiGPR1-149为SsGPR1基因沉默转化子。沉默转化子在PDA培养基上形成的菌核数量及均重与野生型菌株无显著性差异,且菌核均能萌发形成子囊盘,但菌丝生长稠密,生长速度显著下降。在含有H_2O_2的培养基中,SsGPR1基因沉默转化子菌丝生长受到更强的抑制,表明沉默转化子对氧化胁迫更加敏感。SsGPR1基因沉默转化子在活体油菜叶片和拟南芥植株上能引起发病,但病斑面积减小,表明沉默转化子致病力减弱。SsGPR1基因沉默转化子菌丝中的游离脯氨酸含量与野生型菌株相比无显著差异。【结论】SsGPR1与核盘菌的生长和菌丝形态相关,且参与核盘菌对氧化胁迫的抵御及致病过程。
【Objective】The gamma-glutamyl phosphate reductase(GPR) is a key enzyme in fungal proline synthesis pathway.The objective of this study is to silence a GPR-encoded gene SsGPR1 in Sclerotinia sclerotiorum via the RNA interference strategy, research the mycelial growth, sclerotial formation and pathogenicity of the gene-silencing transformants, so as to lay a foundation for revealing the growth, development and pathogenicity of S.sclerotiorum.It also provides important clues for the green prevention and control of Sclerotinia disease.【Method】Homology analysis and phylogenetic tree construction were performed through the BLAST search and MEGA 5.0 software.The real-time RT-PCR was used to detect the expression pattern of SsGPR1 at the different stages of mycelial growth, sclerotial development and germination, and infection processes.The gene silencing vector of SsGPR1 was constructed based on the principle of RNA interference, and the vector was used to transform wild-type strain 1980 by PEG-mediated transformation of protoplasts methods.The gene-silenced strains were identified by real-time RT-PCR.The mycelial morphology, growth rate and sclerotial formation of the gene-silenced strains were observed and the hyphal growth rate under the oxidative stress was measured.The gene-silenced strains were inoculated on Brassica napus leaves and Arabidopsis thaliana plants, and lesion size was observed and measured.The free proline of gene-silenced strains was assayed using the acid ninhydrin method.【Result】SsGPR1 of S.sclerotiorum is 1 454 bp in length and encodes 449 amino acids.SsGPR1 protein contains a GPR domain at amino acid H~(10)-N~(426).SsGPR1 showed high sequence similarity with Botrytis cinerea BC1G_13183(95% identities) and Sclerotinia borealis SBOR_2215 protein(94% identities).The three proteins clustered into a small branch according to the result of phylogenetic tree.SsGPR1 showed high expression level during hyphae growth.The expression level was similar during the different development stages of sclerotia, but it significantly decreased compared with that during the hyphal growth period.The expression level of SsGPR1 increased gradually during the pathogenic period, and it reached the highest at 9 h post inoculation.The SsGPR1 silencing vector, pSIGPR1 was transformed into the wild-type strain of S.sclerotiorum, and the expression level of SsGPR1 in different transformants was detected by real-time RT-PCR.The results showed that SiGPR1-104 and SiGPR1-149 were SsGPR1 gene-silenced transformants.When cultured on PDA medium, SsGPR1 gene-silenced strains had no significant difference with wild-type strain on the number and average dry weight of sclerotia, which can germinate to form apothecium.However, the gene-silenced strains produced denser hyphae and showed a significantly reduce in growth rate.The hyphal growth of SsGPR1 gene-silenced strains was inhibited more strongly when cultured on medium containing H_2O_2, indicating that the gene-silenced strains were more sensitive to oxidative stress.SsGPR1 gene-silenced strains led to small lesions on B.napus leaves and A.thaliana plants, indicating that the pathogenicity of the gene-silenced strains was impaired.The content of proline produced by SsGPR1 gene-silenced strains had no significant difference with wild-type strain.【Conclusion】SsGPR1 is related to hyphal growth and mycelial morphology, and involved in the oxidative stress resistance and pathogenicity of S.sclerotiorum.
【Objective】The gamma-glutamyl phosphate reductase(GPR) is a key enzyme in fungal proline synthesis pathway.The objective of this study is to silence a GPR-encoded gene SsGPR1 in Sclerotinia sclerotiorum via the RNA interference strategy, research the mycelial growth, sclerotial formation and pathogenicity of the gene-silencing transformants, so as to lay a foundation for revealing the growth, development and pathogenicity of S.sclerotiorum.It also provides important clues for the green prevention and control of Sclerotinia disease.【Method】Homology analysis and phylogenetic tree construction were performed through the BLAST search and MEGA 5.0 software.The real-time RT-PCR was used to detect the expression pattern of SsGPR1 at the different stages of mycelial growth, sclerotial development and germination, and infection processes.The gene silencing vector of SsGPR1 was constructed based on the principle of RNA interference, and the vector was used to transform wild-type strain 1980 by PEG-mediated transformation of protoplasts methods.The gene-silenced strains were identified by real-time RT-PCR.The mycelial morphology, growth rate and sclerotial formation of the gene-silenced strains were observed and the hyphal growth rate under the oxidative stress was measured.The gene-silenced strains were inoculated on Brassica napus leaves and Arabidopsis thaliana plants, and lesion size was observed and measured.The free proline of gene-silenced strains was assayed using the acid ninhydrin method.【Result】SsGPR1 of S.sclerotiorum is 1 454 bp in length and encodes 449 amino acids.SsGPR1 protein contains a GPR domain at amino acid H~(10)-N~(426).SsGPR1 showed high sequence similarity with Botrytis cinerea BC1G_13183(95% identities) and Sclerotinia borealis SBOR_2215 protein(94% identities).The three proteins clustered into a small branch according to the result of phylogenetic tree.SsGPR1 showed high expression level during hyphae growth.The expression level was similar during the different development stages of sclerotia, but it significantly decreased compared with that during the hyphal growth period.The expression level of SsGPR1 increased gradually during the pathogenic period, and it reached the highest at 9 h post inoculation.The SsGPR1 silencing vector, pSIGPR1 was transformed into the wild-type strain of S.sclerotiorum, and the expression level of SsGPR1 in different transformants was detected by real-time RT-PCR.The results showed that SiGPR1-104 and SiGPR1-149 were SsGPR1 gene-silenced transformants.When cultured on PDA medium, SsGPR1 gene-silenced strains had no significant difference with wild-type strain on the number and average dry weight of sclerotia, which can germinate to form apothecium.However, the gene-silenced strains produced denser hyphae and showed a significantly reduce in growth rate.The hyphal growth of SsGPR1 gene-silenced strains was inhibited more strongly when cultured on medium containing H_2O_2, indicating that the gene-silenced strains were more sensitive to oxidative stress.SsGPR1 gene-silenced strains led to small lesions on B.napus leaves and A.thaliana plants, indicating that the pathogenicity of the gene-silenced strains was impaired.The content of proline produced by SsGPR1 gene-silenced strains had no significant difference with wild-type strain.【Conclusion】SsGPR1 is related to hyphal growth and mycelial morphology, and involved in the oxidative stress resistance and pathogenicity of S.sclerotiorum.
引文
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[17]TAKAGI H.Proline as a stress protectant in yeast:physiological functions,metabolic regulations,and biotechnological applications.Applied Microbiology&Biotechnology,2008,81(2):211-223.
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[19]杨佳文,赵尊练,张管曲,谢芳琴,姜长岳,张永香,韩晓萍,徐乃林.陕西线辣椒炭疽病原菌的鉴定及生物学特性研究.西北农业学报,2017,26(11):1695-1705.YANG J W,ZHAO Z L,ZHANG G Q,XIE F Q,JIANG C Y,ZHANG Y X,HAN X P,XU N L.Identification and biological characterization of anthrax bacteria in Xianlajiao chili pepper in Shaanxi Province.Acta Agriculturae Boreali-Occidentalis Sinica,2017,26(11):1695-1705.(in Chinese)
[20]许祥明,叶和春,李国凤.脯氨酸代谢与植物抗渗透胁迫的研究进展.植物学通报,2000,17(6):536-542.XU X M,YE H C,LI G F.Progress in synthesis and metabolism of proline and its relationship with osmtolerance of plants.Chinese Bulletin of Botany,2000,17(6):536-542.(in Chinese)
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[22]DELAUNEY A J,HU C A,KISHOR P B,VERMA D P.Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis.Journal of Biological Chemistry,1993,268(25):18673-18678.
[23]赵瑞雪,朱慧森,程钰宏,董宽虎.植物脯氨酸及其合成酶系研究进展.草业科学,2008,25(2):90-97.ZHAO R X,ZHU H S,CHENG Y H,DONG K H.Research progress on proline and its biosynthesis enzymes in plant.Pratacultural Science,2008,25(2):90-97.(in Chinese)
[24]XU L,CHEN W.Random T-DNA mutagenesis identifies a Cu/Zn superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum.Molecular Plant-Microbe Interactions,2013,26(4):431-441.
[25]APOSTOL I,HEINSTEIN P F,LOW P S.Rapid stimulation of an oxidative burst during elicitation of cultured plant cells:role in defense and signal transduction.Plant Physiology,1989,90(1):109-116.
[26]HAREL A,BERCOVICH S,YARDEN O.Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner.Molecular Plant-Microbe Interactions,2006,19(6):682-693.
[27]YU Y,JIANG D H,XIE J T,CHENG J S,LI G Q,YI X H,FU Y P.Ss-Sl2,a novel cell wall protein with PAN modules,is essential for sclerotial development and cellular integrity of Sclerotinia sclerotiorum.PLoS ONE,2012,7(4):e34962.
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[2]BOLAND G J,HALL R.Index of plant hosts of Sclerotinia sclerotiorum.Canadian Journal of Plant Pathology,1994,16(2):93-108.
[3]PURDY L H.Sclerotinia sclerotiorum:history,diseases and symptomatology,host range,geographic distribution,and impact.Phytopathology,1979,69(8):875-880.
[4]YU Y,XIAO J,DU J,YANG Y,BI C,QING L.Disruption of the gene encoding endo-β-1,4-xylanase affects the growth and virulence of Sclerotinia sclerotiorum.Frontiers in Microbiology,2016,7:1787.
[5]POUSSEREAU N,CRETON S,BILLON-GRAND G,RASCLE C,FEVRE M.Regulation of acp1,encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sclerotiorum.Microbiology,2001,147(3):717-726.
[6]KIM K S,MIN J Y,DICKMAN M B.Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development.Molecular Plant-Microbe Interactions,2008,21(5):605-612.
[7]WILLIAMS B,KABBAGE M,KIM H J,BRITT R,DICKMAN M B.Tipping the balance:Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment.PLOS Pathogens,2011,7(6):e1002107.
[8]KABBAGE M,YARDEN O,DICKMAN M B.Pathogenic attributes of Sclerotinia sclerotiorum:switching from a biotrophic to necrotrophic lifestyle.Plant Science,2015,233:53-60.
[9]KABBAGE M,WILLIAMS B,DICKMAN M B.Cell death control:the interplay of apoptosis and autophagy in the pathogenicity of Sclerotinia sclerotiorum.PLoS Pathogens,2013,9(4):e1003287.
[10]朱虹,祖元刚,王文杰,阎永庆.逆境胁迫条件下脯氨酸对植物生长的影响.东北林业大学学报,2009,37(4):86-89.ZHU H,ZU Y G,WANG W J,YAN Y Q.Effect of proline on plant growth under different stress conditions.Journal of Northeast Forestry University,2009,37(4):86-89.(in Chinese)
[11]TROTEL-AZIZ P,NIOGRET M C,DELEU C,BOUCHEREAU A,AZIZ A,LARHER F R.The control of proline consumption by abscisic acid during osmotic stress recovery of canola leaf discs.Physiologia Plantarum,2003,117(2):213-221.
[12]KISHOR P,HONG Z,MIAO G H,HU C,VERMA D.Overexpression of [delta]-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants.Plant Physiology,1995,108(4):1387-1394.
[13]WU G,BAZER F W,BURGHARDT R C,JOHNSON G A,KIM SW,KNABE D A,LI P,LI X,MCKNIGHT J R,SATTERFIELD MC,SPENCER T E.Proline and hydroxyproline metabolism:implications for animal and human nutrition.Amino Acids,2011,40(4):1053-1063.
[14]谢虹,杨兰,李忠光.脯氨酸在植物非生物胁迫耐性形成中的作用.生物技术通报,2011(2):23-27.XIE H,YANG L,LI Z G.The roles of proline in the formation of plant tolerance to abiotic stress.Biotechnology Bulletin,2011(2):23-27.(in Chinese)
[15]NANJO T,KOBAYASHI M,YOSHIBA Y,KAKUBARI Y,YAMAGUCHISHINOZAKI K,SHINOZAKI K.Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana.FEBS Letters,1999,461(3):205-210.
[16]SZéKELY G,ABRAHáM E,CSéPLO A,RIGó G,ZSIGMOND L,CSISZáR J,AYAYDIN F,STRIZHOV N,JáSIK J,SCHMELZERE,KONCZ C,SZABADOS L.Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis.The Plant Journal,2008,53(1):11-28.
[17]TAKAGI H.Proline as a stress protectant in yeast:physiological functions,metabolic regulations,and biotechnological applications.Applied Microbiology&Biotechnology,2008,81(2):211-223.
[18]CHEN C,DICKMAN M B.Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii.Proceedings of the National Academy of Sciences of the United States of America,2005,102(9):3459-3464.
[19]杨佳文,赵尊练,张管曲,谢芳琴,姜长岳,张永香,韩晓萍,徐乃林.陕西线辣椒炭疽病原菌的鉴定及生物学特性研究.西北农业学报,2017,26(11):1695-1705.YANG J W,ZHAO Z L,ZHANG G Q,XIE F Q,JIANG C Y,ZHANG Y X,HAN X P,XU N L.Identification and biological characterization of anthrax bacteria in Xianlajiao chili pepper in Shaanxi Province.Acta Agriculturae Boreali-Occidentalis Sinica,2017,26(11):1695-1705.(in Chinese)
[20]许祥明,叶和春,李国凤.脯氨酸代谢与植物抗渗透胁迫的研究进展.植物学通报,2000,17(6):536-542.XU X M,YE H C,LI G F.Progress in synthesis and metabolism of proline and its relationship with osmtolerance of plants.Chinese Bulletin of Botany,2000,17(6):536-542.(in Chinese)
[21]DELAUNEY A J,VERMA D P S.Proline biosynthesis and osmoregulation in plants.The Plant Journal,1993,4(2):215-223.
[22]DELAUNEY A J,HU C A,KISHOR P B,VERMA D P.Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis.Journal of Biological Chemistry,1993,268(25):18673-18678.
[23]赵瑞雪,朱慧森,程钰宏,董宽虎.植物脯氨酸及其合成酶系研究进展.草业科学,2008,25(2):90-97.ZHAO R X,ZHU H S,CHENG Y H,DONG K H.Research progress on proline and its biosynthesis enzymes in plant.Pratacultural Science,2008,25(2):90-97.(in Chinese)
[24]XU L,CHEN W.Random T-DNA mutagenesis identifies a Cu/Zn superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum.Molecular Plant-Microbe Interactions,2013,26(4):431-441.
[25]APOSTOL I,HEINSTEIN P F,LOW P S.Rapid stimulation of an oxidative burst during elicitation of cultured plant cells:role in defense and signal transduction.Plant Physiology,1989,90(1):109-116.
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