抑制谷胱甘肽合成对大豆胞囊线虫发育的影响
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摘要
为明确谷胱甘肽对大豆胞囊线虫(Heterodera glycines,SCN)发育的影响,利用谷胱甘肽合成抑制剂L-丁硫氨酸-亚砜亚胺(L-Buthionine-sulfoximine,BSO)处理大豆感病品种辽豆15,观察SCN的发育;同时,利用高效液相色谱法(HPLC)和实时荧光定量PCR(qRT-PCR)检测谷胱甘肽含量和合成关键酶基因的表达。结果表明,在接种线虫14d,SCN在辽豆15根系内的发育受到了抑制,二龄幼虫(J2)比例显著高于对照53.86%,四龄幼虫(J4)比例显著低于对照10.54%;但在接种线虫28d时,J4数量与对照比无显著差异。在接种线虫14d,辽豆15根系内谷胱甘肽含量及合成关键酶基因γ-ECS、GSHS和hGSHS的表达量均显著低于对照。BSO有效地抑制了谷胱甘肽合成途径关键酶基因的表达,降低了谷胱甘肽的生物合成产物的含量。在谷胱甘肽缺乏的条件下,大豆胞囊线虫的发育受到了一定程度的抑制。说明谷胱甘肽是参与调控SCN发育的重要因子。
For better understanding the development of soybean cyst nematode( SCN,Heterodera glycines) affected by glutathione,L-Buthionine-sulfoximine( BSO),an inhibitor of glutathione biosynthesis,was used for treating on Liaodou15,a SCN susceptible cultivar,and the development of SCN was examined. The contents and levels of gene expression were detected using qRT-PCR and HPLC,respectively. The results indicated that the development of SCN was inhibited significantly at 14 days post inoculation. Compared with control,the number of juvenile at second stage and juvenile at fourth stage increased to 53. 86% and decreased to 10. 54% respectively;but,the number of J4 showed no difference at 28 days post SCN infection. The expression level of γ-ECS,GSHS and hGSHS were inhibited significantly during BSO treatment at 14 days post SCN infection. The contents of glutathione decreased significantly comparing with control. In summary,the development of SCN was inhibited under glutathione deficiency. And glutathione was a significant factor in development of SCN. These results provided a theoretic support for SCN resistance breeding in future.
For better understanding the development of soybean cyst nematode( SCN,Heterodera glycines) affected by glutathione,L-Buthionine-sulfoximine( BSO),an inhibitor of glutathione biosynthesis,was used for treating on Liaodou15,a SCN susceptible cultivar,and the development of SCN was examined. The contents and levels of gene expression were detected using qRT-PCR and HPLC,respectively. The results indicated that the development of SCN was inhibited significantly at 14 days post inoculation. Compared with control,the number of juvenile at second stage and juvenile at fourth stage increased to 53. 86% and decreased to 10. 54% respectively;but,the number of J4 showed no difference at 28 days post SCN infection. The expression level of γ-ECS,GSHS and hGSHS were inhibited significantly during BSO treatment at 14 days post SCN infection. The contents of glutathione decreased significantly comparing with control. In summary,the development of SCN was inhibited under glutathione deficiency. And glutathione was a significant factor in development of SCN. These results provided a theoretic support for SCN resistance breeding in future.
引文
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[18]刘维志.植物线虫学研究技术[M].沈阳:辽宁科学技术出版社,1995.71-72.
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[21]金京石,沈丽,崔胜云.衍生化法测定山药中还原型谷胱甘肽和总巯基含量[J].食品安全质量检测学报,2013,(4):1 155-1 160.
[22]Foyer C H,Noctor G.Redox regulation in photosynthetic organisms:signaling,acclimation,and practical implications[J].Antioxid Redox Signal,2009,11:861-905.
[23]Bellafiore S,Shen Z,Rosso M N,et al.Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential[J].PLos Pathogens,2008,4:e1000192.
[24]Dubreuil G,Magliano M,Deleury E,et al.Transcriptome analysis of root-knot nematode functions induced in the early stages of parasitism[J].New Phytologist,2007,176:426-436.
[2]Doupnik J R.Soybean production and disease loss estimates for north central United States from 1989 to 1991[J].Plant Disease,1993,77(11):1 170-1 172.
[3]吴海燕,远方,陈立杰,等.大豆胞囊线虫病与大豆抗胞囊线虫机制的研究[J].大豆科学,2001,20(4):285-289.
[4]孙家骥,李爽,朱晓峰,等.胞囊线虫侵染抗感大豆品种后根系谷胱甘肽代谢水平差异研究[J].大豆科学,2016,35(3):468-471.
[5]Rouhier N,Lemaire S D,Jacquot J P.The role of glutathione in photosynthetic organisms:emerging functions for glutaredoxins and glutathionylation[J].Annual Review of Plant Biology,2008,59:143-166.
[6]Ball L,Accotto G P,Bechtold U,et al.Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis[J].Plant Cell,2004,16:2 448-2 462.
[7]Yi H,Ravilious G E,Galant A,et al.From sulfur to homoglutathione:thiol metabolism in soybean[J].Amino Acids,2010,39(4):963-978.
[8]Parisy V,Poinssot B,Owsianowski L,et al.Identification of PAD2 as a gamma-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[J].The Plant Journal,2007,49:159
[9]Schlaeppi K,Bodenhausen N,Buchala A,et al.The glutathione-deficient mutant pad2-1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis[J].The Plant Journal,2008,55:774-786.
[10]Klapheck S.Homoglutathione:isolation,quantification and occurrence in legumes[J].Plant Physiology,1988,74:727-732.
[11]Frendo P,Gallesi D,Turnbull R,et al.Localisation of glutathione and homoglutathione in Medicago truncatula is correlated to a differential expression of genes involved in their synthesis[J].The Plant Journal,1999,17:215-219.
[12]Matamoros M A,Baird L M,Escuredo P R,et al.Stress induced legume root nodule senescence.Physiological,biochemical and structural alterations[J].Plant Physiology,1999,121:97-112.
[13]Frendo P,Harrison J,Norman C,et al.Glutathione and homoglutathione play a critical role in the nodulation process of Medicago truncatula[J].Molecular Plant Microbe Interactions,2005,18:254-259.
[14]Pucciariello C,Innocenti G,Van de Velde W,et al.(Homo)glutathione depletion modulates host gene expression during the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti[J].Plant Physiology,2009,151:1 186-1 196.
[15]Griffith O W,Meister A.Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine(S-n-butyl homocysteine sulfoximine)[J].Journal of Biological Chemistry,1979,254(16):7 558-7 560.
[16]Rosso M N,Dubrana M P,Cimbolini N,et al.Application of RNA interference to root-knot nematode genes encoding esophageal gland roteinsb[J].Molecular Plant Microbe Interactions,2005,18:615-620.
[17]Baldaccicresp F,Chang C,Maucourt M,et al.(Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula[J].PLo S Pathogens,2012,8(1),e1002471.
[18]刘维志.植物线虫学研究技术[M].沈阳:辽宁科学技术出版社,1995.71-72.
[19]Kereszt A,Li D,Indrasumunar A,et al.Agrobacteriumrhizogenes-mediated transformation of soybean to study root biology[J].Nature Protocol.2007,2:948-952.
[20]De Jesus Miranda V,Coelho R R,Viana A A B,et al.Validation of reference genes aiming accurate normalization of q PCR data in soybean upon nematode parasitism and insect attack[J].BMC Research Notes,2013,6(1),196.
[21]金京石,沈丽,崔胜云.衍生化法测定山药中还原型谷胱甘肽和总巯基含量[J].食品安全质量检测学报,2013,(4):1 155-1 160.
[22]Foyer C H,Noctor G.Redox regulation in photosynthetic organisms:signaling,acclimation,and practical implications[J].Antioxid Redox Signal,2009,11:861-905.
[23]Bellafiore S,Shen Z,Rosso M N,et al.Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential[J].PLos Pathogens,2008,4:e1000192.
[24]Dubreuil G,Magliano M,Deleury E,et al.Transcriptome analysis of root-knot nematode functions induced in the early stages of parasitism[J].New Phytologist,2007,176:426-436.