水稻纹枯病菌谷胱甘肽S-转移酶基因的生物信息学及表达分析
|
摘要
通过生物信息学方法及表达分析对水稻纹枯病菌谷胱甘肽S转移酶基因(Rsgst)的功能进行探索。通过水稻纹枯病菌RSIADB基因组数据库查找Rsgst序列,确定该基因在水稻纹枯病菌基因组中的精确位置。利用生物信息学软件预测Rsgst编码蛋白氨基酸序列的基本信息,并使用MEGA 5.0软件构建同源蛋白的系统发育树,最后用qRT-PCR检测Rsgst在菌核发育过程中的基因表达量,同时测定GST的酶活性。结果表明,Rsgst全长1 207 bp,该基因共编码277个氨基酸残基,其编码蛋白分子量为30.90 ku,理论等电点为9.42。该蛋白二级结构中以α-螺旋为主,占41.52%。系统发育树表明,水稻纹枯病菌GST蛋白与担子菌类玉米丝黑穗病菌GST蛋白亲缘关系最近。qRT-PCR结果表明,Rsgst在水稻纹枯病菌菌核发育过程中表达量不断上调,基因最高表达量是在第60小时,为7.64,而Rsgst的最高酶活性是在第5天,其酶活为0.375 U/μg。结果可为预测水稻纹枯病菌中Rsgst基因的功能奠定基础。
The aim of this study is to provide the basic for the exploration of Rsgst genes by bioinformatics and gene expression analysis. Based on the R. solani AG1-IA genome database RSIADB,the sequence of Rsgst gene was found. Basic information of the GST amino acid sequence was predicted by several softwares,and a phylogenetic tree was constructed using MEGA 5. 0 software. qRT-PCR was used to detect the gene expression levels during the progress of sclerotial development of R. solani AG1-IA,and the GST activity was measured simultaneously. The results showed that the full length of Rsgst was 1 207 bp,and the gene encodes 277 amino acid residues. The molecular weight of GST was 30. 90 ku and the theoretical isoelectric point was 9. 42. The secondary structure of this protein was mainly consisted by α-helix which accounted for 41. 52%. The phylogenetic tree showed that the R. solani GST was more closely related to Sporisorium reilianum GST. qRT-PCR indicated that the expression of Rsgst was upregulated during the sclerotial development with the highest gene expression at 60 h,which was 7. 64,and the maximal activity of GST was 0. 375 U/μg at the 5 th day. These results are significant to decipher the functions of Rsgst in R.solani AG1-IA.
The aim of this study is to provide the basic for the exploration of Rsgst genes by bioinformatics and gene expression analysis. Based on the R. solani AG1-IA genome database RSIADB,the sequence of Rsgst gene was found. Basic information of the GST amino acid sequence was predicted by several softwares,and a phylogenetic tree was constructed using MEGA 5. 0 software. qRT-PCR was used to detect the gene expression levels during the progress of sclerotial development of R. solani AG1-IA,and the GST activity was measured simultaneously. The results showed that the full length of Rsgst was 1 207 bp,and the gene encodes 277 amino acid residues. The molecular weight of GST was 30. 90 ku and the theoretical isoelectric point was 9. 42. The secondary structure of this protein was mainly consisted by α-helix which accounted for 41. 52%. The phylogenetic tree showed that the R. solani GST was more closely related to Sporisorium reilianum GST. qRT-PCR indicated that the expression of Rsgst was upregulated during the sclerotial development with the highest gene expression at 60 h,which was 7. 64,and the maximal activity of GST was 0. 375 U/μg at the 5 th day. These results are significant to decipher the functions of Rsgst in R.solani AG1-IA.
引文
[1]Chen X J,Li L L,Zhang Y,et al.Functional analysis of polygalacturonase gene Rs PG2 from Rhizoctonia solani,the pathogen of rice sheath blight[J].European Journal of Plant Pathology,2017,149(2):491-502.
[2]李涛,路雪君,廖晓兰,等.水稻纹枯病的发生及其防治策略[J].江西农业学报,2010,22(9):91-93.
[3]杨迎青,杨媚,兰波,等.水稻纹枯病菌致病机理的研究进展[J].中国农学通报,2014(28):245-250.
[4]张正禹,董文汉,包文静,等.立枯丝核菌AG-1IA对水稻致病力及粗毒素活性的测定方法研究[J].植物病理学报,2017,47(1):82-91.
[5]蔡国雄.水稻化学农药减量控害技术实践与推广[J].农家科技,2017(4):24-25.
[6]苏品.稻鸭种养生态系统抑制水稻纹枯病的发生流行规律及拮抗菌SU8生防潜力研究[D].长沙:湖南农业大学,2012.
[7]Georgiou C D,Patsoukis N,Papapostolou I,et al.Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress[J].Integrative and Comparative Biology,2006,46(6):691-712.
[8]Zhang Z,Wang J Y,Chai R Y,et al.An S-(hydroxymethyl)glutathione dehydrogenase is involved in conidiation and full virulence in the rice blast fungus magnaporthe oryzae[J].PLo S One,2015,10(3):e120627.
[9]王玮玮,唐亮,周文龙,等.谷胱甘肽生物合成及代谢相关酶的研究进展[J].中国生物工程杂志,2014,34(7):89-95.
[10]贾贞,王丹,游松.谷胱甘肽的研究进展[J].沈阳药科大学学报,2009(3):238-242.
[11]Ohnuma T,Anan E,Hoashi R,et al.Dietary diacetylene falcarindiol induces phase 2 Drug-Metabolizing enzymes and blocks Carbon Tetrachloride-Induced hepatotoxicity in mice through suppression of lipid peroxidation[J].Biological&Pharmaceutical Bulletin,2011,34(3):371-378.
[12]Nahar K,Hasanuzzaman M,Alam M M,et al.Roles of exogenous glutathione in antioxidant defense system and methylglyoxal detoxification during salt stress in mung bean[J].Biologia Plantarum,2015,59(4):745-756.
[13]魏涛.真菌谷胱甘肽转移酶(GST)基因家庭的进化与功能分化研究[D].北京:中国科学院大学,2011.
[14]张媛.产黄青霉谷胱甘肽转移酶基因的克隆、表达与功能研究[D].石家庄:河北师范大学,2007.
[15]Cho Y W,Park E H,Fuchs J A,et al.A second stressinducible glutathione S-transferase gene from Schizosaccharomyces pombe[J].Biochimica et Biophysica acta,2002,1574(3):399-402.
[16]Veal E A,Toone W M,Jones N,et al.Distinct roles for glutathione S-transferases in the oxidative stress response in Schizosaccharomyces pombe[J].Journal of Biological Chemistry,2002,277(38):35523-35531.
[17]胡晓明.家蚕雄蛾触角特异谷胱甘肽-S-转移酶Bm GSTD4的基因克隆、表达及功能研究[D].重庆:西南大学,2012.
[18]Mcgoldrick S,O'sullivan S M,Sheehan D.glutathione transferase-like proteins encoded in genomes of yeasts and fungi:insights into evolution of a multifunctional protein superfamily[J].FEMS Microbiology Letters,2005,242(1):1-12.
[19]马萧萧.酵母谷胱甘肽转移酶Gtt2和甲硫氨酸亚砜还原酶Mxr1的结构与功能研究[D].合肥:中国科学技术大学,2011.
[20]马立功,孟庆林,张匀华,等.向日葵谷胱甘肽-S-转移酶基因的克隆及抗病功能研究[J].中国油料作物学报,2015,37(5):635-643.
[21]戴瀚洋,孙洋,柏立新,等.亚致死浓度甲维盐胁迫对甜菜夜蛾幼虫解毒酶系活力及其相关基因表达量的影响[J].棉花学报,2015,27(2):149-158.
[22]Fujimiya T,Kobayashi Y,Aoki T,et al.Effect of the GSTM1 null genotype on glutathione S-transferase(GST)activity in patients with non-viral liver tumors[J].Showa Univ J Med Sci,2017,28(2):113-121.
[23]肖广侠,徐文远,贾磊,等.悬浮物对褐牙鲆肌肉抗氧化酶活性及相关基因表达的影响[J].海洋科学进展,2016,34(4):542-552.
[24]Feng S J,Shu C W,Wang C J,et al.Survival of rhizoctonia solani AG-1 IA,the causal agent of rice sheath blight,under different environmental conditions[J].Journal of Phytopathology,2017,165(1):44-52.
[25]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.
[26]秦勇.细胞内ROS水平改变对细胞活性及相关信号传导途径的影响[D].杭州:浙江大学,2012.
[27]李素娟,邢艳刚,薛星晨,等.氟对小鼠睾丸谷胱甘肽抗氧化系统的影响[J].畜牧兽医学报,2017,48(5):954-962.
[28]张雪,陶磊,乔晟,等.谷胱甘肽转移酶在植物抵抗非生物胁迫方面的角色[J].中国生物工程杂志,2017,37(3):92-98.
[29]Zhou Y L,LüY P,Qiu L N,et al.Mechanism of sodium nitroprusside-induced apoptosis in K562 cell line[J].Journal of Experimental Hematology,2005,13(6):983.
[2]李涛,路雪君,廖晓兰,等.水稻纹枯病的发生及其防治策略[J].江西农业学报,2010,22(9):91-93.
[3]杨迎青,杨媚,兰波,等.水稻纹枯病菌致病机理的研究进展[J].中国农学通报,2014(28):245-250.
[4]张正禹,董文汉,包文静,等.立枯丝核菌AG-1IA对水稻致病力及粗毒素活性的测定方法研究[J].植物病理学报,2017,47(1):82-91.
[5]蔡国雄.水稻化学农药减量控害技术实践与推广[J].农家科技,2017(4):24-25.
[6]苏品.稻鸭种养生态系统抑制水稻纹枯病的发生流行规律及拮抗菌SU8生防潜力研究[D].长沙:湖南农业大学,2012.
[7]Georgiou C D,Patsoukis N,Papapostolou I,et al.Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress[J].Integrative and Comparative Biology,2006,46(6):691-712.
[8]Zhang Z,Wang J Y,Chai R Y,et al.An S-(hydroxymethyl)glutathione dehydrogenase is involved in conidiation and full virulence in the rice blast fungus magnaporthe oryzae[J].PLo S One,2015,10(3):e120627.
[9]王玮玮,唐亮,周文龙,等.谷胱甘肽生物合成及代谢相关酶的研究进展[J].中国生物工程杂志,2014,34(7):89-95.
[10]贾贞,王丹,游松.谷胱甘肽的研究进展[J].沈阳药科大学学报,2009(3):238-242.
[11]Ohnuma T,Anan E,Hoashi R,et al.Dietary diacetylene falcarindiol induces phase 2 Drug-Metabolizing enzymes and blocks Carbon Tetrachloride-Induced hepatotoxicity in mice through suppression of lipid peroxidation[J].Biological&Pharmaceutical Bulletin,2011,34(3):371-378.
[12]Nahar K,Hasanuzzaman M,Alam M M,et al.Roles of exogenous glutathione in antioxidant defense system and methylglyoxal detoxification during salt stress in mung bean[J].Biologia Plantarum,2015,59(4):745-756.
[13]魏涛.真菌谷胱甘肽转移酶(GST)基因家庭的进化与功能分化研究[D].北京:中国科学院大学,2011.
[14]张媛.产黄青霉谷胱甘肽转移酶基因的克隆、表达与功能研究[D].石家庄:河北师范大学,2007.
[15]Cho Y W,Park E H,Fuchs J A,et al.A second stressinducible glutathione S-transferase gene from Schizosaccharomyces pombe[J].Biochimica et Biophysica acta,2002,1574(3):399-402.
[16]Veal E A,Toone W M,Jones N,et al.Distinct roles for glutathione S-transferases in the oxidative stress response in Schizosaccharomyces pombe[J].Journal of Biological Chemistry,2002,277(38):35523-35531.
[17]胡晓明.家蚕雄蛾触角特异谷胱甘肽-S-转移酶Bm GSTD4的基因克隆、表达及功能研究[D].重庆:西南大学,2012.
[18]Mcgoldrick S,O'sullivan S M,Sheehan D.glutathione transferase-like proteins encoded in genomes of yeasts and fungi:insights into evolution of a multifunctional protein superfamily[J].FEMS Microbiology Letters,2005,242(1):1-12.
[19]马萧萧.酵母谷胱甘肽转移酶Gtt2和甲硫氨酸亚砜还原酶Mxr1的结构与功能研究[D].合肥:中国科学技术大学,2011.
[20]马立功,孟庆林,张匀华,等.向日葵谷胱甘肽-S-转移酶基因的克隆及抗病功能研究[J].中国油料作物学报,2015,37(5):635-643.
[21]戴瀚洋,孙洋,柏立新,等.亚致死浓度甲维盐胁迫对甜菜夜蛾幼虫解毒酶系活力及其相关基因表达量的影响[J].棉花学报,2015,27(2):149-158.
[22]Fujimiya T,Kobayashi Y,Aoki T,et al.Effect of the GSTM1 null genotype on glutathione S-transferase(GST)activity in patients with non-viral liver tumors[J].Showa Univ J Med Sci,2017,28(2):113-121.
[23]肖广侠,徐文远,贾磊,等.悬浮物对褐牙鲆肌肉抗氧化酶活性及相关基因表达的影响[J].海洋科学进展,2016,34(4):542-552.
[24]Feng S J,Shu C W,Wang C J,et al.Survival of rhizoctonia solani AG-1 IA,the causal agent of rice sheath blight,under different environmental conditions[J].Journal of Phytopathology,2017,165(1):44-52.
[25]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.
[26]秦勇.细胞内ROS水平改变对细胞活性及相关信号传导途径的影响[D].杭州:浙江大学,2012.
[27]李素娟,邢艳刚,薛星晨,等.氟对小鼠睾丸谷胱甘肽抗氧化系统的影响[J].畜牧兽医学报,2017,48(5):954-962.
[28]张雪,陶磊,乔晟,等.谷胱甘肽转移酶在植物抵抗非生物胁迫方面的角色[J].中国生物工程杂志,2017,37(3):92-98.
[29]Zhou Y L,LüY P,Qiu L N,et al.Mechanism of sodium nitroprusside-induced apoptosis in K562 cell line[J].Journal of Experimental Hematology,2005,13(6):983.