Sequence Characterization and Differential Expression Analysis of a Aryl Alcohol Oxidase Gene vvaao1 from Volvariella volvacea

doi:10.13560/j.cnki.biotech.bull.1985.2018-0166

Abstract

Abstract: A gene encoding aryl alcohol oxidase named vvaao1 was obtained from straw mushroom Volvariella volvacea. To reveal the role of vvaao1 during substrate degradation and fruiting-body development in V. volvacea,the structure,sequence characters and expression profile of vvaao1 were analyzed. ZOOM software was used to confirm the accuracy of gene sequence and analyze the gene structure. The protein sequence was submitted to Expasy ProtParam for physicochemical properties prediction,SignalP server for signal peptide prediction,TargetP server for sub-cellular localization prediction,and the Phyre 2 server for three dimensional（3D）structure analysis. The MEGA 5.1 was used for multiple sequence alignment and phylogenetic tree analysis. Digital gene expression profiling（DGE）,real time fluorescent quantitative PCR（RT-qPCR）and iTRAQ methods were used to determine the expression pattern of vvaao1. Results showed that the full sequence of vvaao1 covered 3 091 bp,containing 9 exons and 8 introns,and the open reading frame length was 1803bp,encoded a protein with 600 amino acids. Bioinformatic analysis showed that the VvAAO1 contained a secretory signal peptide,and located in secretory pathway. The 3D structures predicted showed that VvAAO1 consisted with 20 α-helices and 19 β-strands,and had 48% identity with model protein 3FIM. The phylogenetic tree showed that VvAAO1 of V. volvacea belonged to the AAO class of GMC superfamily,and had the closest relationship with AAO in Pleurotus eryngii and P. pulmonarius. The analysis of DGE,RT-qPCR and iTRAQ results showed that the expression value of vvaao1 in H1521 were 51.0,49.8 and 2.13 times as much as the sum of two homokaryons,respectively. In addition,the gene expression of vvaao1 in primordia showed significant higher than other fruiting-body samples. VvAAO1 is a secretory protein,and its high level expression may be beneficial for fruiting-body formation.

Key words: brown-rot fungi, primordium formation, aromatic alcohol oxidase, gene structure, synergistic gene expression effect

YAN Jun-jie, TONG Zong-jun, LIU Yuan-yuan, ZHANG Lei, ZHANG Yun, XIE Bao-gui. Sequence Characterization and Differential Expression Analysis of a Aryl Alcohol Oxidase Gene vvaao1 from Volvariella volvacea[J]. Biotechnology Bulletin, 2018, 34(4): 107-114.

References

[1] Tao Y, Xie B, Yang Z, et al.Identification and expression analysis of a new glycoside hydrolase family 55 exo-β-1, 3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development[J]. Gene, 2013, 527(1):154-160.
[2] 李正鹏, 余昌霞, 李巧珍. 草菇工厂化栽培原材料研究进展[J]. 农业工程技术:温室园艺, 2017, 37(19):67-70.
[3] 关雁桃, 罗国杨, 董炳炎, 等. 变废为宝--广东菇农利用药渣栽培草菇技术[J]. 当代蔬菜, 2005(10):27-27.
[4] 朱刚, 吴林, 陈明杰, 等. 草菇锰过氧化物酶编码基因生物信息学分析及其转录水平和酶活性的测定[J]. 菌物学报, 2013, 32(5):919-927.
[5] 杨晔. 木质素降解酶研究进展[J]. 农业工程, 2014(4):48-51.
[6] Goswami P, Chinnadayyala SSR, Chakraborty M, et al.An overview on alcohol oxidases and their potential applications[J]. Applied Microbiology and Biotechnology, 2013, 97(10):4259-4275.
[7] Hernández-Ortega A, Ferreira P, Martínez AT.Fungal aryl-alcohol oxidase:a peroxide-producing flavoenzyme involved in lignin degradation[J]. Applied Microbiology and Biotechnology, 2012, 93(4):1395-1410.
[8] Varela E, Böckle B, Romero A, et al.Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius[J]. Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology, 2000, 1476(1):129-138.
[9] Chen B, Gui F, Xie B, et al.Composition and expression of genes encoding carbohydrate-active enzymes in the straw-degrading mushroom Volvariella volvacea[J]. PLoS One, 2013, 8(3):e58780.
[10] 刘靖宇, 江玉姬, 谢宝贵, 等. 应用iTRAQ 结合 2D LC-MS/MS 技术分析草菇同核和异核菌丝蛋白质组[J]. 食用菌学报, 2012, 19(1):1-6.
[11] Zhang Z, Lin H, Ma B.ZOOM Lite:next-generation sequencing data mapping and visualization software[J]. Nucleic Acids Research, 2010, 38:W743-W748.
[12] Yan JJ, Zhang L, Wang RQ, et al.The sequence characteristics and expression models reveal superoxide dismutase involved in cold response and fruiting body development in Volvariella volvacea[J]. International Journal of Molecular Sciences, 2016, 17(1):34.
[13] Carro J, Serrano A, Ferreira P, et al.Fungal aryl-alcohol oxidase in lignocellulose degradation and bioconversion[M]//GuptaVK. Microbial Enzymes in Bioconversions of Biomass. Switzerland:Springer International Publishing, 2016:301-322.
[14] Tamura K, Peterson D, Peterson N, et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28(10):2731-2739.
[15] 刘朋虎, 谢宝贵, 邓优锦, 等. 草菇gpi 基因结构及其异核体表达协同增效作用[J]. 食用菌学报, 2011, 18(4):1-5.
[16] Livak KJ, Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J]. Methods, 2001, 25:402-408.
[17] Ma B, Zhang K, Hendrie C, et al.PEAKS:Powerful Software for Peptide De Novo Sequencing by MS/MS[J]. Rapid Communications in Mass Spectrometry, 2003, 17(20):2337-2342.
[18] 严俊杰, 郭丽羡, 赵静静, 等. 草菇谷胱甘肽 S-转移酶编码基因(vv-gto1)的序列分析及其差异表达[J], 2014, 54(10):1171-1177.
[19] 王圣铕, 廖伟, 黄千慧, 等. 生理生化指标与草菇开伞时间的相关性分析[J]. 基因组学与应用生物学, 2016, 35(4):970-978.
[20] 朱坚. 食用菌品种特性与栽培[M]. 福州:福建科学技术出版社, 2011.
[21] 陆娜, 周祖法, 袁卫东, 等. 不同草菇菌株的室内床架式栽培试验[J]. 食药用菌, 2010, 18(3):28-29.
[22] 吴林, 朱刚, 陈明杰, 等. 草菇基因组中 11 个漆酶同源基因的生物信息学分析及铜离子对其表达的影响[J]. 菌物学报, 2014, 33(2):323-333.
[23] 闫强, 王安建, 王高尚, 等. 全球生物质能资源评价[J]. 中国农学通报, 2009, 25(18):466-470.
[24] Ruiz-Dueñas FJ, Martínez ÁT.Microbial degradation of lignin:how a bulky recalcitrant polymer is efficiently recycled in nature and how we can take advantage of this[J]. Microbial Biotechnology, 2009, 2(2):164-177.
[25] 吴茵, 陈敏, 郭倩. 刺芹侧耳芳基醇氧化酶的分离纯化及其酶学性质[J]. 菌物学报, 2016, 35(6):705-713.
[26] 项林平, 刘小芸, 田强强, 等. 棉铃虫核型多角体病毒几丁质酶的原核表达及其协同增效作用[J]. 西北农林科技大学学报:自然科学版, 2011, 39(2):87-91.
[27] 王莹莹, 任相亮, 姜伟丽, 等. 鳢肠乙醇粗提物与 Cry1Ca 的协同增效及对甜菜夜蛾 SeABCC1-SeABCC3 mRNA 表达量的影响[J]. 中国生物防治学报, 2017, 33(4):446-453.
[28] Meng L, Yan J, Xie B, et al.Genes encoding FAD-binding proteins in Volvariella volvacea exhibit differential expression in homokaryons and heterokaryons[J]. Microbiological Research, 2013, 168(8):533-546.
[29] 刘朋虎, 邓优锦, 江玉姬, 等. 草菇TPI 基因的克隆, 结构及其在同核, 异核菌株中的表达量[J]. 福建农林大学学报:自然科学版, 2013(1):63-67.
[30] 刘朋虎, 谢宝贵, 邓优锦, 等. 草菇磷酸果糖激酶(PFK)基因克隆, 结构及不同菌株中表达量分析[J]. 菌物学报, 2013, 32(2):253-260.