指状青霉cyt b5和cyt b5r基因克隆及与cyp51A表达

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.024

摘要/Abstract

摘要： 为探究指状青霉细胞色素b₅（Cyt b₅）与细胞色素b₅还原酶（Cyt b₅r）在细胞色素P450 CYP51A电子传递方面的功用,研究了指状青霉CYP51A与Cyt b₅-Cyt b₅r共表达机制;并检测了其对于cyp51A基因表达水平的影响。通过转录组分析筛选并PCR克隆获得了cyt b₅与 cyt b₅r基因,分别命名为HS-Pdcyt b₅和HS-Pdcyt b₅r。以多基因串联克隆载体pPICZαA为骨架构建了指状青霉共表达质粒ppbrA（pPIC-Pdcyp51A-cyt b₅-cyt b₅r）;电转化法将重组质粒ppbrA导入毕赤酵母X-33中。qRT-PCR分析结果显示,CYP51A与Cyt b₅-Cyt b₅r共表达后,其基因表达水平升高54%-97%,并维持较长时间（48-72 h）。表明Cyt b₅-Cyt b₅r系统可将电子高效转移给CYP51A,从而增强cyp51A基因的转录表达。从指状青霉中克隆表达HS-PdCyt b₅和HS-PdCyt b₅r蛋白,并通过共表达的方式研究cyp51A基因的功能尚为首次报道。

关键词: 指状青霉CYP51A, 共表达, 细胞色素b5, 细胞色素b5还原酶, 毕赤酵母X-33

Abstract: In order to investigate the role of Cytochrome b₅（Cyt b₅）and Cytochrome b₅ reductase（Cyt b₅r）in the electron transport of cytochrome P450 CYP51A in Penicillium digitatum,the co-expression mechanism of CYP51A and Cyt b₅-Cyt b₅r in P. digitatum was studied,and its effects on the expression of gene cyp51A was detected. By analyzing and screening transcriptome as well as PCR cloning,gene cyt b5 and cyt b₅r were acquired and designated as HS-Pdcyt b₅ and HS-Pdcyt b₅r. Further,a co-expressed plasmid vector ppbrA（pPIC-Pdcyp51A-cyt b₅-cyt b₅r）was constructed successfully using multiple-gene series cloning vector pPICZαA. This recombinant plasmid ppbrA was transformed into Pichia pastoris X-33 by electroporation. Analysis by qRT-PCR revealed that after CYP51A was co-expressed with Cyt b₅-Cyt b₅r,the expression level of cyp51A increased 54%-97% and it remained in a long period（48-72 h）. This indicated that the Cyt b₅-Cyt b₅r complex was capable of transferring electrons to CYP51A,which thus enhanced the transcript expression level of cyp51A. This is the first report regarding cloning and expressing HS-PdCyt b₅ and HS-PdCyt b₅r proteins from P. digitatum,and explore the function of P. digitatum’s gene cyp51A by co-expressing system.

Key words: Penicillium digitatum CYP51A, co-expression, cytochrome b5, cytochrome b5 reductase, Pichia pastoris X-33

秦婷婷, 耿辉, 王胜强, 牛玉慧, 伍志刘德立. 指状青霉cyt b₅和cyt b₅r基因克隆及与cyp51A表达[J]. 生物技术通报, 2016, 32(9): 179-188.

QIN Ting-ting, GENG Hui, WANG Sheng-qiang, NIU Yu-hui, WU Zhi, LIU De-li. Cloning of Gene cyt b₅ and cyt b₅r and Their Co-expression with cyp51A in Penicillium digitatum[J]. Biotechnology Bulletin, 2016, 32(9): 179-188.

参考文献

[1] Sanborn RC, Williams CM. The cytochrome system in the Cecropia Silkworm, with special reference to the properties of a new component[J]. Journal of General Physiology, 1950, 33（5）：579-588.
[2] Hildebrandt A, Estabrook RW. Evidence for the participation of cytochrome b 5 in hepatic microsomai mixed-function oxidation reactions[J]. Archives of Biochemistry and Biophysics, 1971, 143（1）：66-79.
[3] Shimakata T, Mihara K, Sato R. Reconstitution of hepatic microsomal stearoyl-coenzyme A desaturase system from solubilized components[J]. Journal of Biological Chemistry, 1972, 72（5）：1163-1174.
[4] Keyes SR, Alfano JA, Jansson I, et al. Rat liver microsomal elongat-ion of fatty acids. Possible involvement of cytochrome b 5 [J]. Journal of Biological Chemistry, 1979, 254（16）：7778-7784.
[5] Storbeck KH, Swart AC, Goosen P, et al. Cytochrome b 5 ：Novel roles in steroidogenesis[J]. Molecular and Cellular Endocrinology, 2013, 371（1-2）：87-99.
[6] Mokashi V, Li L, Porter TD. Cytochrome b 5 reductase and cytochrome b 5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test[J]. Archives of Biochemistry and Biophysics, 2003, 412（1）：147-152.
[7] Kurian JR, Bajad SU, Miller JL, et al. Trepanier LA. NADH cytochrome b 5 reductase and cytochrome b 5 catalyze the microsomal reduction of xenobiotic hydroxylamines and amidoximes in humans[J]. Journal of Pharmacology and Experimental Therapeutics, 2004, 311（3）：1171-1178.
[8] Li L, Porter TD. Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase-null mice[J]. Journal of Biochemical and Molecular Toxicology, 2009, 23（5）：357-363.
[9] Lamb DC, Kelly DE, Manning NJ, et al. Biodiversity of the P450 catalytic cycle：Yeast cytochrome b 5 /NADH cytochrome b 5 reductase complex efficiently drives the entire sterol 14-demethylation（CYP51）reaction[J]. Febs Letters, 1999, 462 （3）：283-288.
[10] Troncoso C, Carcamo J, Hedden P, et al. Influence of electron transport proteins on the reactions catalyzed by Fusarium Fujikuroi gibberellin monooxygenases[J]. Phytochemistry, 2008, 69（3）：672-683.
[11] Subramanian V, Doddapaneni H, Syed K, et al. P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium：gene transcription, heterologous expression, and activity analysis on the purified proteins[J]. Current Microbiology, 2010, 61（4）：306-314.
[12] Dong MS, Lee SB, Kim HJ. Co-expression of human cytochrome b 5 increases expression of cytochrome P450 3A4 in Escherichia coli by stabilizing mRNA[J]. Protein Expression and Purification, 2013, 89（1）：44-50.
[13] Syed K, Kattamuri C, Thompson TB, et al. Cytochrome b 5 reductase-cytochrome b 5 as an active P450 redox enzyme system in Phanerochaete chrysosporium：atypical properties and in vivo evidence of electron transfer capability to CYP63A2[J]. Archives of Biochemistry and Biophysics, 2011, 509（1）：26-32.
[14] Liu J, Wang SQ, Qin TT, et al. Whole transcriptome analysis of Penicillium digitatum strains treatmented with prochloraz reveals their drug-resistant mechanisms[J]. BMC Genomics, 2015, 16 （1）：855.
[15] Bridges A, Gruenke L, Chang YT, et al. Identification of the binding site on cytochrome P450 2B4 for cytochromeb 5 and cytochrome P450 reductase[J]. Journal of Biological Chemistry, 1998, 273（27）：17036-17049.
[16] Clarke TA, Im SC, Bidwai A, et al. The role of the length and sequence of the linker domain of cytochrome b 5 in stimulating cytochrome P450 2B4 catalysis[J]. Journal of Biological Chemistry, 2004, 279（35）：36809-36818.
[17] Zhang H, Im SC, Waskell L. Cytochrome b 5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4[J]. Journal of Biological Chemistry, 2007, 282（41）：29766-29776.
[18] Zhang M, Le Clair SV, Huang R, et al. Insights into the role of substrates on the interaction between cytochrome b 5 and cytochrome P450 2B4 by NMR[J]. Scientific Reports, 2015, 5：8392.
[19] Zhang H, Gao N, Liu TT, et al. Effect of cytochrome b 5 content on the activity of polymorphic CYP1A2, 2B6, and 2E1 in human liver microsomes[J]. PLoS ONE, 2015, 10（6）：e0128547.
[20] Vishwesh M, Li L, Todd DP. Cytochrome b 5 reductase and cytochrome b 5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test[J]. Archives of Biochemistry and Biophysics, 2003, 412（1）：147-152.

指状青霉cyt b₅和cyt b₅r基因克隆及与cyp51A表达

Cloning of Gene cyt b₅ and cyt b₅r and Their Co-expression with cyp51A in Penicillium digitatum

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 13

编辑推荐

Metrics

本文评价

[1]	韩惠, 张舰, 任宇红. 短链脱氢酶Lvchun的分子改造及其在氯霉胺合成中的应用[J]. 生物技术通报, 2023, 39(4): 81-92.
[2]	周敏雅, 陆睿, 张婷, 袁婷婷, 卢瑶瑶, 严坤宁, 袁玉国, 成勇. 重组人SOD1/3转基因山羊的制备及表达产物的检测[J]. 生物技术通报, 2019, 35(5): 85-92.
[3]	黄勤勤, 王慧梅, 梁玲, 黄钦耿, 吴松刚, 黄建忠. lysC定点突变及lysC、asdA串联表达对谷氨酸棒杆菌L-苏氨酸积累的影响[J]. 生物技术通报, 2019, 35(2): 93-100.
[4]	李骥璇, 余磊, 李京美, 郑桂兰, 王洪钟. S-亚胺还原酶和葡萄糖脱氢酶共表达系统的构建及手性胺的合成[J]. 生物技术通报, 2019, 35(1): 105-111.
[5]	全聪, 张静, 吴辉, 李志敏, 叶勤. CydDC蛋白的表达及对谷胱甘肽运输的影响[J]. 生物技术通报, 2016, 32(11): 255-260.
[6]	王慧, 崔云风, 刘岩, 史吉平, 赵志军, 王绍明. MntH与含锰过氧化氢酶共表达基因工程菌的构建与发酵优化[J]. 生物技术通报, 2015, 31(9): 183-189.
[7]	钟开新, 陈丽芝, 李阳源. 共表达内质网响应蛋白HAC1对α-半乳糖苷酶在毕氏酵母中表达的影响[J]. 生物技术通报, 2014, 0(7): 184-189.
[8]	汪涛, 余辉, 梅钧, 李兴暖, 周小鸥, 赵志军. 猪链球菌2型MRP和EF双基因共表达重组腺病毒载体的构建[J]. 生物技术通报, 2013, 0(8): 130-134.
[9]	黄云雁;黎明;刘萌;孙昕;周丽颖;路福平;. 赖氨酸-尸胺反向转运蛋白cadB基因谷氨酸棒杆菌表达载体的构建及转化[J]. , 2012, 0(08): 94-100.
[10]	黄朋;范升龙;凌敏;贺菽嘉;周素芳;. 肝癌相关抗原SMP30重组基因的构建、表达及分子伴侣共表达系统的探索[J]. , 2012, 0(04): 170-175.
[11]	赵宏峰;边英男;黄伟达;. 利用重组大肠杆菌生产双乙酰[J]. , 2011, 0(01): 165-169.
[12]	陈降华;刘忠华;李文平;. 猪Ⅱ型圆环病毒ORF2与猪GM-CSF基因重组腺病毒共表达及免疫效果分析[J]. , 2009, 0(12): 124-128.
[13]	项海涛;杨彬;温峰琴;胡永浩;柳纪省;. 双效表达载体的构建及其U6启动子的功能效率鉴定[J]. , 2009, 0(04): 82-85.