柑橘全爪螨PcSOD3的异源表达及其重组酶的抗氧化活性

doi:10.3864/j.issn.0578-1752.2016.24.006

摘要/Abstract

摘要： 【目的】笔者课题组前期研究发现，柑橘全爪螨在极端温度、杀螨剂、重金属和紫外线胁迫下，其体内PcSOD3表达显著上调，暗示PcSOD3在柑橘全爪螨应对不良环境胁迫的过程中起着至关重要的作用。为了进一步明确PcSOD3的生理功能，本研究开展了该基因的异源表达及重组酶生化特性的分析工作。【方法】构建pET28a-PcSOD3重组表达质粒，并在大肠杆菌中实现异源表达。随后利用镍柱亲和层析法分离纯化PcSOD3重组酶，采用Western blot对纯化蛋白进行验证。使用WST-1法分析PcSOD3重组酶的抗氧化活性，测定不同反应体系pH及不同前处理温度下PcSOD3重组酶的活性。采用氯化铬、叔丁基过氧化氢和过氧化氢异丙苯3种氧化性药剂，诱导大肠杆菌细胞内产生氧化应激反应，并利用Kirby-Bauer纸片琼脂糖扩散法测定上述3种药剂对过表达PcSOD3大肠杆菌的抑制效果。【结果】在大肠杆菌BL21（DE3）菌株中成功表达PcSOD3，研究摸索出PcSOD3重组蛋白最适诱导表达条件：诱导表达温度为18℃，摇床转速为160 r/min，当OD值达0.6时加入IPTG并使其终浓度为0.4 mmol·L^-1，诱导时间为18 h。Western blot验证结果表明所纯化重组蛋白即为PcSOD3重组酶，重组蛋白大小为25.3 kD。WST-1法测得PcSOD3重组酶活性在pH=7.0的反应体系中最高，约为47.3 U/mg protein，而在前处理温度为25℃时，PcSOD3重组酶活性最高为40.2 U/mg protein。利用K-B纸片琼脂糖扩散法进行了药敏试验，结果显示氯化铬对过量表达PcSOD3大肠杆菌产生的抑菌圈均显著小于对空载体对照产生的抑菌圈，通过测量发现抑菌圈较对照缩小近20%；在150 mmol·L^-1浓度下叔丁基过氧化氢对过量表达PcSOD3大肠杆菌产生的抑菌圈与对照相比缩小约25%；而浓度为200 mmol·L^-1的过氧化氢异丙苯对过量表达PcSOD3大肠杆菌产生的抑菌圈与对照相比则缩小约15%。【结论】成功在大肠杆菌中实现了PcSOD3的功能性表达，并纯化获得了PcSOD3重组蛋白。明确了PcSOD3重组酶最适反应pH及最适前处理温度等生化特性，WST-1法证明PcSOD3重组蛋白在离体条件下具有显著的抗氧化活性，而K-B纸片琼脂糖扩散法则证明过表达PcSOD3的大肠杆菌抗氧化能力显著增强，说明PcSOD3具有抗氧化功能。研究结果进一步揭示PcSOD3在柑橘全爪螨抗氧化损伤过程中具有重要作用。

关键词: 柑橘全爪螨, 超氧化物歧化酶, 生化特性, 重组蛋白, 药敏试验

Abstract: 【Objective】In the author’s previous studies, it was found that PcSOD3 is up-regulated after exposure to thermal stress, acaricide abamectin, heavy metal and UV-B ultraviolet irradiation, which suggested that PcSOD3 played an important role in the response of P. citri to various adverse environmental stress. In order to further elucidate the physiological functions of PcSOD3, the biochemical characteristics of its recombinant protein expressed in Escherichia coli was investigated. 【Method】The pET28a-PcSOD3 expression plasmid for the heterologous expression in E. coli was constructed. The recombinant protein was purified by using a Ni⁺affinity chromatography column, and confirmed by the Western blot analysis. The enzymatic activity was measured using a SOD total activity assay kit. Moreover, the effects of various pH values of the reaction system and different temperatures for preincubation on the recombinant enzyme were tested using the same kit. Using the Kirby-Bauer disc diffusion assay method, the diameters of inhibition zones of the E. coli overexpressing PcSOD3 were measured by exposing to chromic chloride, t-butylhydroperoxide and cumene hydroperoxide. 【Result】 PcSOD3 was successfully expressed in the BL21 (DE3) strain of E. coli. The best condition for the induction and expression of the recombinant protein was obtained, under which cells were cultured at 18℃ and 160 r/min, IPTG was added to a final concentration of 0.4 mmol·L^-1, the inducing time continued for 18 h. The resulted PcSOD3 recombinant protein was further confirmed by the Western blot analysis, with the molecular weight of 25.3 kD. Moreover, the PcSOD3 recombinant protein was active in the WST-1 activity assay system. Based on the assay system, it was found that PcSOD3 recombinant protein was mostly active (activity was about 47.3 U/mg protein) when the pH of the reaction system was 7.0, while it was mostly active (activity was about 40.2 U/mg protein) when the preincubation temperature was 25℃. As indicated by the Kirby-Bauer test, the diameters of inhibition zones of the E. coli overexpressing PcSOD3 which were exposed to chromic chloride (CdCl₂), were about 20% smaller than the control. Similarly, overexpression of PcSOD3 in E. coli had significantly reduced the inhibition zone (around 25%) caused by exposure to t-butylhydroperoxide at the concentration of 150 mmol·L^-1, compared to the control. Meanwhile, E. coli expressing PcSOD3 showed 15% reduced inhibition when exposed to cumene hydroperoxide.【Conclusion】PcSOD3 was functionally expressed in E. coli. Purified PcSOD3 recombinant protein was obtained. Furthermore, the biochemical characteristics of the recombinant enzyme such as the optimal pH and preincubation temperature was determined. The recombinant enzyme showed a significant antioxidation activity in vitro by WST-1 assay method. The results of Kirby-Bauer disc diffusion assays showed that the E. coli overexpressing PcSOD3 had a significantly elevated tolerance to the oxidative stress, which has proven that PcSOD3 has the antioxidant function. Results of the experiment further revealed that PcSOD3 played a crucial role in the tolerance of P. citri to oxidative stress.

Key words: Panonychus citri, superoxide dismutase (SOD), biochemical characteristics, recombinant protein, Kirby-Bauer test

蒋红波，冯英财，刘世火，王进军. 柑橘全爪螨PcSOD3的异源表达及其重组酶的抗氧化活性[J]. 中国农业科学, 2016, 49(24): 4735-4744.

JIANG Hong-bo, FENG Ying-cai, LIU Shi-huo, WANG Jin-jun. Heterologous Expression of PcSOD3 from Panonychus citri and the Anti-oxidant Activity of Its Recombinant Enzyme[J]. Scientia Agricultura Sinica, 2016, 49(24): 4735-4744.

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参考文献

[1] Liao C Y, Zhang K, Niu J Z, Ding T B, Zhong R, Xia W K, Dou W, Wang J J. Identification and characterization of seven glutathione S-transferase genes from citrus red mite, Panonychus citri (McGregor). International Journal of Molecular Sciences, 2013, 14(12): 24255-24270.

[2] Campbell B, Nelson R, Ebel R, Dozier W, Adrian J, Hockema B. Fruit quality characteristics that affect consumer preferences for satsuma mandarins. HortScience, 2004, 39(1): 1664-1669.

[3] Fadamiro H Y, Xiao Y, Hargroder T, Nesbitt M, Umeh V, Childers C C. Seasonal occurrence of key arthropod pests and associated natural enemies in Alabama Satsuma citrus. Environmental Entomology, 2008, 37(2): 555-567.

[4] Niu J Z, Hull-Sanders H, Zhang Y X, Lin J Z, Dou W, Wang J J. Biological control of arthropod pests in citrus orchards in China. Biological Control, 2014, 68: 15-22.

[5] Cheng L L, Nechols J R, Margolies D C, Campbell J F, Yang P S, Chen C C, Lu C T. Efficacy of the predator Mallada basalis (Neuroptera: Chrysopidae) on Tetranychus kanzawai and Panonychus citri (Acari: Tetranychidae) at different predator:prey release ratios. Journal of Asia-Pacific Entomology, 2012, 15(1): 142-146.

[6] Hu J F, Wang C F, Wang J, You Y, Chen F. Monitoring of resistance to spirodiclofen and five other acaricides in Panonychus citri collected from Chinese citrus orchards. Pest Management Science, 2010, 66(9): 1025-1030.

[7] Zhang K, Niu J Z, Ding T B, Dou W, Wang J J. Molecular characterization of two carboxylesterase genes of the citrus red mite, Panonychus citri (Acari: Tetranychidae). Archives of Insect Biochemistry and Physiology, 2013, 82(4): 213-226.

[8] Cho Y S, Choi B N, Kim K H, Kim S K, Kim D S, Bang I C, Nam Y K. Differential expression of Cu/Zn superoxide dismutase mRNA during exposures to heavy metals in rockbream (Oplegnathus fasciatus). Aquaculture, 2006, 253(1/4): 667-679.

[9] Zhang G H, Liu H, Wang J J, Wang Z Y. Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of the predatory mite, Neoseiulus cucumeris (Acari: Phytoseiidae). Experimental and Applied Acarology, 2014, 64(1): 73-85.

[10] Yang L H, Huang H, Wang J J. Antioxidant responses of citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), exposed to thermal stress. Journal of Insect Physiology, 2010, 56(12): 1871-1876.

[11] Dalton T P, Shertzer H G, Puga A. Regulation of gene expression by reactive oxygen. Annual Review of Pharmacology and Toxicology, 1999, 39(1): 67-101.

[12] El-Shenawy N S. Effects of insecticides fenitrothion, endosulfan and abamectin on antioxidant parameters of isolated rat hepatocytes. Toxicology in Vitro, 2010, 24(4): 1148-1157.

[13] Meng J Y, Zhang C Y, Zhu F, Wang X P, Lei C L. Ultraviolet light-induced oxidative stress: Effects on antioxidant response of Helicoverpa armigera adults. Journal of Insect Physiology, 2009, 55(6): 588-592.

[14] Jia H H, Sun R J, Shi W N, Yan Y, Li H, Guo X Q, Xu B H. Characterization of a mitochondrial manganese superoxide dismutase gene from Apis cerana cerana and its role in oxidative stress. Journal of Insect Physiology, 2014, 60(1): 68-79.

[15] 李成成, 杨维仁, 胥保华, 冯倩倩. 意大利蜜蜂生长发育适宜蛋白供给水平及其对幼虫抗氧化活性的影响. 中国农业科学, 2011, 44(22): 4714-4720.

Li C C, Yang W R, Xu B H, Feng Q Q. Optimal protein levels required and their effects on larval antioxidation of Apis mellifera ligustica Spinola. Scientia Agricultura Sinica, 2011, 44(22): 4714-4720. (in Chinese)

[16] 马义, 霍新北, 马民, 付荣恕. 高效氯氰菊酯对德国小蠊超氧化物歧化酶诱导作用研究. 寄生虫与医学昆虫学报, 2005, 12(1): 37-40.

Ma Y, Huo X B, Ma M, Fu R S. Induction of high effect cypermethrin on superoxide dismutase of Blattella germanica. Acta Parasitologica et Medica Entomologica Sinica, 2005, 12(1): 37-40. (in Chinese)

[17] Gao X M, Jia F X, Shen G M, Jiang H Q, Dou W, Wang J J. Involvement of superoxide dismutase in oxidative stress in the oriental fruit fly, Bactrocera dorsalis: molecular cloning and expression profiles. Pest Management Science, 2013, 69(12): 1315-1325.

[18] 冯从经, 戴华国, 武淑文. 褐飞虱高温条件下应激反应及体内保护酶系活性的研究. 应用生态学报, 2001, 12(3): 409-413.

Feng C J, Dai H G, Wu S W. Stress response of Nilaparvata lugens at high temperature and activities of its protective enzyme systems. Chinese Journal of Applied Ecology, 2001, 12(3): 409-413. (in Chinese)

[19] Jung I, Kim T Y, Kim-Ha J. Identification of Drosophila SOD3 and its protective role against phototoxic damage to cells. FEBS Letters, 2011, 585(12): 1973-1978.

[20] Goulielmos G N, Arhontaki K, Eliopoulos E, Tserpistali K, Tsakas S, Loukas M. Drosophila Cu, Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli. Biochemical and Biophysical Research Communications, 2003, 308(3): 433-438.

[21] Feng Y C, Liao C Y, Xia W K, Jiang X Z, Shang F, Yuan G R, Wang J J. Regulation of three isoforms of SOD gene by environmental stresses in citrus red mite, Panonychus citri. Experimental and Applied Acarology, 2015, 67(1): 49-63.

[22] Bauer A W, Kirby W M M, Sherris J C, Turck M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 1966, 45(4): 493-496.

[23] Gao X L, Li J M, Wang Y L, Jiu M, Yan G H, Liu S S, Wang X W. Cloning, expression and characterization of mitochondrial manganese superoxide dismutase from the whitefly, Bemisia tabaci. International Journal of Molecular Sciences, 2013, 14(1): 871-887.

[24] Tang T, Huang D W, Zhou C Q, Li X, Xie Q J, Liu F S. Molecular cloning and expression patterns of copper/zinc superoxide dismutase and manganese superoxide dismutase in Musca domestica. Gene, 2012, 505(2): 211-220.

[25] Gräslund S, Nordlund P, Weigelt J, Hallberg B M, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J R, dhe-Paganon S, Park H, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim S-H, Rao Z H, Shi Y Y, Terwilliger T C, Kim C-Y, Hung L-W, Waldo G S, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman J L, Stevens R C, Lesley S A, Wilson L A, Joachimiak A, Collart F, Dementieva L, Donnelly M I, Eschenfeldt W H, Kim Y C, Stols L, Wu R Y, Zhou M, Burley S K, Emtage J S, Sauder J M, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo S C, Bonanno J B, Fiser A, Swaminathan S, Studier F W, Chance M R, Sali A, Acton T B, Xiao R, Zhao L, Ma L C, Hunt J F, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho C K, Wang D Y, Wang H, Jiang M, Montelione G T, Stuart D I, Owens R J, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus K C. Protein production and purification. Nature Methods, 2008, 5(2): 135-146.

[26] Selleck W, Tan S. Recombinant protein complex expression in E. coli//Current Protocols in Protein Science, 2008, Chapter 5: 5.21.1-5.21.21.

[27] Ferrer-Miralles N, Saccardo P, Corchero J L, Xu Z K, García-Fruitós E. General introduction: recombinant protein production and purification of insoluble proteins. Methods in Molecular Biology, 2015, 1258: 1-24.

[28] 付楠霞, 翟枫, 姜鸣, 吕淑敏, 张雅林. 锯角豆芫菁法尼基焦磷酸合酶的cDNA克隆、序列分析及原核表达. 中国农业科学, 2014, 47(2): 273-283.

Fu N X, ZHAI F, Jiang M, Lü S M, Zhang Y L. cDNA cloning, sequence analysis and prokaryotic expression of farnesyl pyrophosphate synthase from Epicauta gorhami Marseul (Coleoptera: Meloidae). Scientia Agricultura Sinica, 2014, 47(2): 273-283. (in Chinese)