果桑肥大性菌核病菌多聚半乳糖醛酸酶基因(CsPG1)的克隆及功能分析
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摘要
多聚半乳糖醛酸酶(polygalacturonase,PG)是一种细胞壁结构蛋白,可以催化果胶分子多聚α-(1,4)-聚半乳糖醛酸的裂解,使细胞壁结构解体,导致果实软化。本文采用q RT-PCR方法,分析不同生长阶段菌丝侵染的油菜叶片中多聚半乳糖醛酸酶基因的表达模式。结果表明:(1)采用RT-PCR从果桑肥大性菌核病菌(Ciboria shiraiana)菌核中扩增多聚半乳糖醛酸酶基因c DNA,其全长为1143 bp,命名为CsPG1(Gen Bank登录号为KR296662),编码380个氨基酸残基。根据侵染油菜叶片的表达量,确认CsPG1为基因家族的主效基因;(2)将CsPG1基因连接p ET28a(+)载体并转化大肠杆菌E.coli BL21(DE3),获得包涵体形式,但对底物(聚半乳糖醛酸)没有活性的蛋白;(3)最适CsPG1包涵体溶解的尿素浓度6 mol L~(–1),采用缓慢稀释低温复性的方法对重组蛋白CsPG1进行了重折叠复性试验,经高亲和力的Ni-NTA树脂纯化后为得到单一条带,获得可溶性蛋白,复性后的多聚半乳糖醛酸酶比活力为5.02 U mg–1;(4)生物试验表明,CsPG1蛋白能够加速嘉陵40(Morus atropurpurea Roxb.)桑椹的成熟和软化,推测该蛋白与肥大性菌核病菌对桑椹的侵染有关。这一结果揭示了不同果桑品种对菌核病敏感性的差异,为果桑生产中菌核病的防控提供了分子证据。
Polygalacturonase(PG) is a kind of cell wall structural proteins that can catalyes the decompocition of alpha-(1,4)-polymer of galacturonia acid, which makes the cell wall structure disintegration and fruit softening. In the paper, we analyzed the expression patterns of PG genes in the leaf infection by the hypha of C. shiraiana at different growth stages by using q RT-PCR methods. The results showed that the c DNA of CsPG1 gene was amplified from the sclerotia of C. shiraiana by RT-PCR, namely CsPG1(Gen Bank accession number: KR296662) with 1143 bp of full length, encoding 380 amino acid residues. On the basis of the highest level of gene expression in the process of infecting rape leaf, the main gene of the CsPG gene family was confirmed. CsPG1 was cloned into p ET-28a(+) vector and expressed in E. coli BL21(DE3). The recombinant CsPG1 protein was expressed in the form of inclusion bodies without activity towards polygalaturonic acid. The optimal urea concentration for dissolving CsPG1 inclusion bodies was 6 mol L–1, CsPG1 was renaturated by dilution gradiently at low temperature. We sorted out single band after purified by High-Affinity Ni-NTA Resin, and obtained soluble protein. The specific activity of renatured CsPG1 was 5.02 U mg–1. The result of biological test showed that the recombinant protein accelerated the fruit maturity and softening of Jialing 40(Morus atropurpurea Roxb.). Therefor, we speculated that the CsPG1 protein is related to the infection of C. shiraiana to mulberry fruits. The result reveals the sensitivity difference among mulberry varieties to sclerotial disease, providing the molecular evidence for preventing and controlling the sclerotial disease in mulberry cultivation.
Polygalacturonase(PG) is a kind of cell wall structural proteins that can catalyes the decompocition of alpha-(1,4)-polymer of galacturonia acid, which makes the cell wall structure disintegration and fruit softening. In the paper, we analyzed the expression patterns of PG genes in the leaf infection by the hypha of C. shiraiana at different growth stages by using q RT-PCR methods. The results showed that the c DNA of CsPG1 gene was amplified from the sclerotia of C. shiraiana by RT-PCR, namely CsPG1(Gen Bank accession number: KR296662) with 1143 bp of full length, encoding 380 amino acid residues. On the basis of the highest level of gene expression in the process of infecting rape leaf, the main gene of the CsPG gene family was confirmed. CsPG1 was cloned into p ET-28a(+) vector and expressed in E. coli BL21(DE3). The recombinant CsPG1 protein was expressed in the form of inclusion bodies without activity towards polygalaturonic acid. The optimal urea concentration for dissolving CsPG1 inclusion bodies was 6 mol L–1, CsPG1 was renaturated by dilution gradiently at low temperature. We sorted out single band after purified by High-Affinity Ni-NTA Resin, and obtained soluble protein. The specific activity of renatured CsPG1 was 5.02 U mg–1. The result of biological test showed that the recombinant protein accelerated the fruit maturity and softening of Jialing 40(Morus atropurpurea Roxb.). Therefor, we speculated that the CsPG1 protein is related to the infection of C. shiraiana to mulberry fruits. The result reveals the sensitivity difference among mulberry varieties to sclerotial disease, providing the molecular evidence for preventing and controlling the sclerotial disease in mulberry cultivation.
引文
[1]Cook B J,Clay R P,Bergmann C W,Albersheim P,Darvill A G.Fungal polygalacturonase exhibit different substrate degradation patterns and differ in their susceptibilities to polygalacturonase inhibiting proteins.Appl Environ Microbiol,1999,65:1596–1602
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[3]Hamann T.Plant cell wall integrity maintenance as an essential component of biotic stress response mechanisms.Front Plant Sci,2012,3:77–82
[4]Esquerre Tugaye M T,Boudard G,Dumas B.Cell wall degrading enzymes,inhibitory proteins,and oligosaccharides participate in the molecular dialogue between plants and pathogens.Plant Physiol Biochem,2000,38:157–163
[5]Claudia M,Giuliano D,Giulia D,Vittorio V.Doriano L.Isolation heterologous expression and characterization of an endopolygalactumnase produced by the phytopathogen Burkholderia cepacia.Protein Expres Pufif,2007,54:300–308
[6]Dellapenna D,Lashbrook C C,Toenjes K,Giovannoni J J,Fischer R L,Bennett A B.Polygalacturonase isozymes and pectin depolymerization in transgenic rin tomato fruit.Plant Physiol,1990,94:1882–1886
[7]Ali Z M,Chin L H,Lazan H.A comparative study on wall degrading enzymes,pectin modifications and softening during ripening of selected tropical fruits.Plant Sci,2004,167:317–327
[8]Choi J H,Seongkoo L.Pectic substances associated with woolliness of peaches.J Korean Soc Hort Sci,1999,40:574–576
[9]Bonghi C,Pagni S,Vidrih R.Cell wall hydrolases and amylase in kiwifruit softening.Postharvest Biol Technol,1997,9:19–29
[10]Huber D J,ódonoghue E M.Polyuronides in avocado and tomato fruits exhibit markedly different patterns of molecular weight downshifts during ripening.Plant Physiolol,1993,102:473–480
[11]Kutsunai S Y,Lin A C,Percival F W,Laties G G,Christoffersen R E.Ripeningrelated polygalacturonase c DNA from avocado.Plant Physiol,1993,103:289–290
[12]Huber D J.Strawberry fruit softening:the potential roles of polyuronides and hemicellulose.J Food Sci,2000,49:1310–1315
[13]Villarreal N M,Rosli H G,Maroinez G A,Civello P M.Polygalacturonase activity and expression of related genes during ripening of strawberry cultivars with contrasting fruit firmness.Postharvest Biol Technol,2008,47:141–150
[14]Bonghi C,Rascio N,Ramina A,Casadoro G.Cellulase and polygalacturonase involvement in the abscission of leaf and explants of peach.Plant Mol Biol,1992,20:839–848
[15]Meakin P J,Roberts J A.Anatomical and biochemical changes associated with the induction of oilseed rape pod dehiscence by Dasineura brassicae.Ann Bot,1991,67:193–197
[16]Pressey R.Polygalacturonase in tree pollens.Phytochemistry,1991,30:1753–1755
[17]LüR H,Zhao A H,Li J,Liu C Y,Wang C H,Wang X L,Wang X H,Pei R C,Lu C,Yu M D.Screening,cloning and expression analysis of a cellulase derived from the causative agent of hypertrophy sorosis scleroteniosis Ciboria shiraiana.Gene,2015,565:221–227
[18]方中达.植病研究方法.北京:中国农业出版社,1998.pp123–124Fang Z D.Research Methods in Phytopathology.Beijing:China Agriculture Press,1998.pp 123–124(in Chinese)
[19]吕蕊花,金筱耘,赵爱春,吉洁,刘长英,李军,蒲龙,鲁成,余茂德.果桑肥大性菌核病菌和油菜菌核病菌的交叉侵染、生物学特性及遗传关系.作物学报,2015,41:42–48LüR H,Jin X Y,Zhao A C,Ji J,Liu C Y,Li J,Pu L,Lu C,Yu M D.Cross infection,biological characteristics and genetic relationship between pathogens of hypertrophy sorosis sclerotenisis from mulberry and sclerotinia stem rot from oilseed rape.Acta Agron Sin,2015,41:42–48(in Chinese with English abstract)
[20]Livak K J,Schmittgen T D.Analysis of relative gene expression data using realtime quantitative PCR and the 2(-delta delta C(T))method.Methods,2001,25,402–408
[21]Schoner R G,Ellis L F,Schoner B E.Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone.Nat Biotechno1,1985,3:151–154
[22]Marston F A O,Lowe P A,Doel M T.Purfication of calf prochymosin(prorennin)synthesize in Eschesized coli.Nat Biothechnol,1984,2:800–804
[23]Lindsay H.A colorimetric estimation of reducing sugars in potatoes with 3,5-dinitrosalicylic acid.Potato Res,1973,16:176–179
[24]Liu C Y,Zhao A C,Zhu P P,Li J,Han L,Wang X L,Fan W,LüR H,Wang C H,Wang X H,Lu C,Yu M D.Characterization and expression of Genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.Plo S One,2015,10:e0122081
[25]Zafer D B,Roger R,George G,Khachatourians D,Hegedus D.Factors governing the regulation of Sclerotinia sclerotiorum cutinase A and polygalacturonase during different stages of infection.Can J Microbiol,2012,58:605–616
[26]Kasza Z,Vagv?lgyi C,Fèvre M,Cotton P.Molecular characterization and in planta detection of Sclerotinia sclerotiorum endopolygalacturonase genes.Curr Microbiol,2004,48:208–213
[27]Cotton P,Rascle C,Fevre M.Characterization of PG2,an early endo PG produced by Sclerotinia sclerotiorum,expressed in yeast.FEMS Microbiol Lett,2002,213:239–244
[28]Sella L,Tomassini A,Ovidio R D,Favaron F.Expression of two Sclerotinia sclerotiorum endo PG genes correlates with endopolygalacturonase activity during Glycine max colonization.J Plant Pathol,2005,87:199–205
[29]Lumsden D.Pectolytic enzymes of Sclerotinia sclerotiorum and their location in infected bean.Can J Bot,1976,54:2630–2641
[30]Dawson D W,Melton L D,Watkins C B.Cell wall changes in nectarines:solubilization and depolymentation of pectic and neutral polyments during ripening and in mealy fruit.Plant Physiol,1992,100:1203–1210
[2]Alkorta I,Garbisu C,Llama M,Serra L J.Industrial applications of pectic enzymes:a review.Proc Biochem,1998,33:21–28
[3]Hamann T.Plant cell wall integrity maintenance as an essential component of biotic stress response mechanisms.Front Plant Sci,2012,3:77–82
[4]Esquerre Tugaye M T,Boudard G,Dumas B.Cell wall degrading enzymes,inhibitory proteins,and oligosaccharides participate in the molecular dialogue between plants and pathogens.Plant Physiol Biochem,2000,38:157–163
[5]Claudia M,Giuliano D,Giulia D,Vittorio V.Doriano L.Isolation heterologous expression and characterization of an endopolygalactumnase produced by the phytopathogen Burkholderia cepacia.Protein Expres Pufif,2007,54:300–308
[6]Dellapenna D,Lashbrook C C,Toenjes K,Giovannoni J J,Fischer R L,Bennett A B.Polygalacturonase isozymes and pectin depolymerization in transgenic rin tomato fruit.Plant Physiol,1990,94:1882–1886
[7]Ali Z M,Chin L H,Lazan H.A comparative study on wall degrading enzymes,pectin modifications and softening during ripening of selected tropical fruits.Plant Sci,2004,167:317–327
[8]Choi J H,Seongkoo L.Pectic substances associated with woolliness of peaches.J Korean Soc Hort Sci,1999,40:574–576
[9]Bonghi C,Pagni S,Vidrih R.Cell wall hydrolases and amylase in kiwifruit softening.Postharvest Biol Technol,1997,9:19–29
[10]Huber D J,ódonoghue E M.Polyuronides in avocado and tomato fruits exhibit markedly different patterns of molecular weight downshifts during ripening.Plant Physiolol,1993,102:473–480
[11]Kutsunai S Y,Lin A C,Percival F W,Laties G G,Christoffersen R E.Ripeningrelated polygalacturonase c DNA from avocado.Plant Physiol,1993,103:289–290
[12]Huber D J.Strawberry fruit softening:the potential roles of polyuronides and hemicellulose.J Food Sci,2000,49:1310–1315
[13]Villarreal N M,Rosli H G,Maroinez G A,Civello P M.Polygalacturonase activity and expression of related genes during ripening of strawberry cultivars with contrasting fruit firmness.Postharvest Biol Technol,2008,47:141–150
[14]Bonghi C,Rascio N,Ramina A,Casadoro G.Cellulase and polygalacturonase involvement in the abscission of leaf and explants of peach.Plant Mol Biol,1992,20:839–848
[15]Meakin P J,Roberts J A.Anatomical and biochemical changes associated with the induction of oilseed rape pod dehiscence by Dasineura brassicae.Ann Bot,1991,67:193–197
[16]Pressey R.Polygalacturonase in tree pollens.Phytochemistry,1991,30:1753–1755
[17]LüR H,Zhao A H,Li J,Liu C Y,Wang C H,Wang X L,Wang X H,Pei R C,Lu C,Yu M D.Screening,cloning and expression analysis of a cellulase derived from the causative agent of hypertrophy sorosis scleroteniosis Ciboria shiraiana.Gene,2015,565:221–227
[18]方中达.植病研究方法.北京:中国农业出版社,1998.pp123–124Fang Z D.Research Methods in Phytopathology.Beijing:China Agriculture Press,1998.pp 123–124(in Chinese)
[19]吕蕊花,金筱耘,赵爱春,吉洁,刘长英,李军,蒲龙,鲁成,余茂德.果桑肥大性菌核病菌和油菜菌核病菌的交叉侵染、生物学特性及遗传关系.作物学报,2015,41:42–48LüR H,Jin X Y,Zhao A C,Ji J,Liu C Y,Li J,Pu L,Lu C,Yu M D.Cross infection,biological characteristics and genetic relationship between pathogens of hypertrophy sorosis sclerotenisis from mulberry and sclerotinia stem rot from oilseed rape.Acta Agron Sin,2015,41:42–48(in Chinese with English abstract)
[20]Livak K J,Schmittgen T D.Analysis of relative gene expression data using realtime quantitative PCR and the 2(-delta delta C(T))method.Methods,2001,25,402–408
[21]Schoner R G,Ellis L F,Schoner B E.Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone.Nat Biotechno1,1985,3:151–154
[22]Marston F A O,Lowe P A,Doel M T.Purfication of calf prochymosin(prorennin)synthesize in Eschesized coli.Nat Biothechnol,1984,2:800–804
[23]Lindsay H.A colorimetric estimation of reducing sugars in potatoes with 3,5-dinitrosalicylic acid.Potato Res,1973,16:176–179
[24]Liu C Y,Zhao A C,Zhu P P,Li J,Han L,Wang X L,Fan W,LüR H,Wang C H,Wang X H,Lu C,Yu M D.Characterization and expression of Genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.Plo S One,2015,10:e0122081
[25]Zafer D B,Roger R,George G,Khachatourians D,Hegedus D.Factors governing the regulation of Sclerotinia sclerotiorum cutinase A and polygalacturonase during different stages of infection.Can J Microbiol,2012,58:605–616
[26]Kasza Z,Vagv?lgyi C,Fèvre M,Cotton P.Molecular characterization and in planta detection of Sclerotinia sclerotiorum endopolygalacturonase genes.Curr Microbiol,2004,48:208–213
[27]Cotton P,Rascle C,Fevre M.Characterization of PG2,an early endo PG produced by Sclerotinia sclerotiorum,expressed in yeast.FEMS Microbiol Lett,2002,213:239–244
[28]Sella L,Tomassini A,Ovidio R D,Favaron F.Expression of two Sclerotinia sclerotiorum endo PG genes correlates with endopolygalacturonase activity during Glycine max colonization.J Plant Pathol,2005,87:199–205
[29]Lumsden D.Pectolytic enzymes of Sclerotinia sclerotiorum and their location in infected bean.Can J Bot,1976,54:2630–2641
[30]Dawson D W,Melton L D,Watkins C B.Cell wall changes in nectarines:solubilization and depolymentation of pectic and neutral polyments during ripening and in mealy fruit.Plant Physiol,1992,100:1203–1210