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[1]徐忠新,王红,赵奎军,等.大豆蚜内共生菌groEL克隆及表达水平分析[J].大豆科学,2015,34(06):964-971.[doi:10.11861/j.issn.1000-9841.2015.06.0964]
　XU Zhong-xin,WANG Hong,ZHAO Kui-jun,et al.Molecular Cloning and Analysis of Expression Levels of Buchnera groEL of Aphis glycines(Hemiptera: Aphididae)[J].Soybean Science,2015,34(06):964-971.[doi:10.11861/j.issn.1000-9841.2015.06.0964]

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大豆蚜内共生菌groEL克隆及表达水平分析

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第34卷期数: 2015年06期页码: 964-971 栏目: 出版日期: 2015-12-25

Title:: Molecular Cloning and Analysis of Expression Levels of Buchnera groEL of Aphis glycines(Hemiptera: Aphididae)

作者:: 徐忠新; 王红; 赵奎军; 韩岚岚; 东北农业大学农学院，黑龙江哈尔滨 150030

Author(s):: XU Zhong-xin; WANG Hong; ZHAO Kui-jun; HAN Lan-lan; College of Agriculture, Northeast Agricultural University, Harbin 150030, China

关键词:: 大豆蚜; 大豆花叶病毒; 内共生菌; 病毒结合蛋白基因; 荧光定量 PCR

Keywords:: Aphis glycines; soybean mosaic virus; Buchnera; GroEL; Real-time quantitative PCR

DOI:: 10.11861/j.issn.1000-9841.2015.06.0964

文献标志码:: A

摘要:: 为了解大豆蚜（Aphis glycines Matsumura）传播的大豆花叶病毒（soybean mosaic virus, SMV）与其体内共生菌产生的病毒结合蛋白(GroEL)之间的作用机制，运用RT-PCR技术克隆得到大豆蚜内共生菌groEL基因全序列，把该基因与pET21b载体重组后进行原核表达，经Ni-Agarose His亲和层析得到纯化的蛋白，并利用荧光定量PCR技术分析了饲毒不同时间、不同龄期大豆蚜内共生菌groEL基因表达量的变化。序列分析表明:大豆蚜内共生菌groEL基因全长序列为1 647 bp，编码548个氨基酸，推测蛋白分子量和pI值分别为69 kDa和5.24，成功构建重组载体pET21b.GroEL进行原核表达，Western-blot鉴定确定为目的蛋白，蛋白可溶性分析发现重组蛋白为包涵体。随饲食SMV时间的延长大豆蚜内共生菌groEL的表达量显著增加，且无翅成虫与有翅成虫内共生菌groEL的表达量显著高于其它4个若虫期。大豆蚜内共生菌groEL基因能在原核细胞中稳定、正确表达，并且大豆蚜在饲食SMV后会诱导其内共生菌groEL的表达，从而增加SMV爆发流行的可能性。

Abstract:: In order to probe into the mechanism underlying interaction between propagation of mosaic virus (soybean mosaic virus, SMV) by ?Aphis glycines and its Buchnera-producing GroEL protein. The full-length groEL sequence was amplified from the Buchnera of Aphis glycines by RT-PCR technique, and the gene was recombined into pET21b and expressed in prokaryotic expression system.Meanwhile purified recombinant protein was obtained by Ni-Agarose His affinity chromatography, the content of SMV in Aphis glycinesat different time and the changes of expression levels of groEL gene after feeding SMV were analyzed using Real-time quantitative PCR technology.The sequence analysis showed that the full-length Buchnera groEL sequence of Aphis glycines is 1 647 bp in length encoding 548 amino acid residues with the predicted molecular weight 69 kDa and pI 5.24.The built recombinant vector pET21b-GroEL was expressed in prokaryotic expression system，and the recombinant protein was determinded as the target protein using western-blot analysis. The Buchnera groEL gene expression levels of Aphis glycines was increased significantly with the extension of time of feeding SMV, the expression level of Buchnera groEL gene expression content in wingless and winged adults were significantly higher than the other four nymphs periods. All these results above showed that Buchneragro EL gene of Aphis glycines was expressed stably and correctly in E.coli, and the expression of Buchneragro EL was induced in Aphis glycines after feeding on SMV, which increases the possibility of SMV spread in soybean field.

参考文献/References:

［1］Liu J, Wu K M, Hopper K R,et al. Population dynamics of Aphis glycines (Homoptera: Aphididae)and its natural enemies in soybean in Northern China［J］. Annals of the Entomological Society of America, 2004, 97(2): 235-239.

［2］郭井泉, 张明厚.大豆花叶病毒(SMV)主要介体及其传毒效率研究［J］. 大豆科学, 1989, 8(1): 55-62.(Guo J Q, Zhang M H.Study on the important of soybean mosaic virus and their tansmission efficiencies［J］. Soybean Science, 1989, 8(1): 55-62.)

［3］Hohn T.Plant virus transmission from the insect point of view［J］. American Academy of Sciences,2007, 104(46):17905-17906.

［4］王素云, 暴祥致, 孙雅杰, 等.大豆蚜虫对大豆生长和产量影响的试验［J］. 大豆科学, 1996, 15(3): 243-247. (Wang S Y, Bao X Z， Sun Y J, et al. Studyon effect of population dinamics of soybean aphis（Aphisglycines）on both growth and yield soybean［J］.Soybean Science,1996, 15(3): 243-247)

［5］Wu T L, Ma X H, Yao L M, et al. Identification of soybean resources of resistance to aphids［J］ Agricultural Sciences in China, 1996, 8(8): 979-984

［6］Ishikawa H. Characterization of protein species synthesized in vivo and vitro-by an aphid endosymbiont［J］.Insect Biochemistry and Molecular Biology, 1984, 14(4): 417-425

［7］Power A G. Insect transmission of plant viruses: a constraint on virus variability［J］ Current Opinion in Plant Biology, 2000, 3(4): 336-340.

［8］Gray S M, Banerjee N. Mechanisms of arthropod transmission of plant and animal viruses［J］.Microbiology and Molecular Biology Reviews, 1999, 63(1): 128-148.

［9］谭周进, 肖启明, 谢丙炎, 等.昆虫内共生菌及其传病毒相关groEL蛋白［J］.生物技术通讯, 2004, 15(5): 498-501. (Tan Z J, Xiao Q M, Xie B Y, et al.Insect endosymbionts and their protein GroEL relation to transmitting viruses［J］.Letters in Biotechnology, 2004,15(5): 498-501.)

［10］Morin S, Ghanim M, Zei D M, et al.A groEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus［J］.Virology, 1999, 256(1): 75-84.

［11］吴云锋, 林林, 崔晓峰. 麦二叉蚜体内病毒结合蛋白基因的克隆和原核表达［J］. 病毒学报, 2002, 18(3): 275-278.(Wu Y F, Lin L, Cui X F, et al.Cloning and prokaryotic expression of virus binding protein gene from Schizaphisgraminum［J］.Chinese Journal of Virology, 2002, 18(3): 275-278.)?

［12］Akad F, Dotan N, Czosnek H.Trapping of tomato yellow leaf curl virus (TYLCV) and other plant viruses with a groEL homologue from the whitefly Bemisiatabaci［J］. Archives of Virology, 2004, 149(8): 1481-1497.

［13］吴云锋, 崔晓锋, 林林,等.禾谷缢管蚜体内的病毒结合蛋白基因的克隆与原核表达［J］.微生物学报, 2002, 42(4): 448-452.(Wu Y F, Cui X F, Lin L, et al.Cloning and prokaryotic expression of virus binding protein gene fromschizaphisgraminum［J］.Chinese Journal of Virology, 2002, 42(4): 448-452.)

［14］林林, 吴云锋, 崔晓峰.玉米蚜体内参与传毒的内共生菌groEL基因的克隆和原核表达［J］.中国病毒学, 2003,18 (1):54-57. (Lin L, Wu Y F, Cui X F.Cloning and prokaryotic expression of groEL gene of endosybioticbacterium from Rhopalosiphum maize［J］Virologicasinica, 2003, 18 (1): 54-57.)

［15］崔晓峰, 吴云锋, 林林,等. 桃蚜体内与病毒结合的共生菌Buchnera GroEL基因的克隆和序列分析［J］.中国病毒学, 2001,17(1): 69-72.(Cui X F, Wu Y F, Lin L, et al. Cloning and sequence analysis of Buchnera GroEL gene from endosybiotic bacterium of Myzuspersicae［J］ Virologica Sinica, 2001,17(1): 69-72.)

［16］Sambrook J, Russell D W.Molecular cloning: A laboratory manual［M］.3rd ed New York:Cold Spring Harbor Laboratoy Press, 2011

［17］王渭霞, 罗举, 赖凤香,等水稻褐飞虱内生共生细菌Arsenophonus的鉴定和系统分析［J］.昆虫学报, 2010, 53(6): 647-654. (Wang W X, Luo J, Lai F X, et al.Identification and phylogenetic analysis of symbiotic bacteria Arsenophonus from the rice brown planthopper, N ilaparvatalugens(St.1/4l) (Homoptera:Delphacidae)［J］.Acta Entomologica Sinica, 2010, 53(6): 647-654.)

［18］谈忠鸣, 李志峰, 吴斌,等.基于GroEL基因的实时荧光定量PCR检测恙虫病东方体方法的建立及评价［J］.中国人兽共患病学报, 2014, 30(11): 1121-1124.(Tan Z M, Li Z F, Wu B, et al. Establishment and evaluation of real-time PCR assay for detection of Orientia tsutsugamushi based on the GroEL gene［J］. Chinese Journal of Zoonoses, 2014, 30(11): 1121-1124.)

［19］Livak K J, Schmittgen T D. Analysis of relative gene expressiondata using real-time quantitative PCR and the 2－ ΔΔC.T. method［J］. Methods, 2001, 25(4): 402-408.

［20］Wan F, Zhang G, Liu S, et al.Invasive mechanism andmanagement strategy of Bemisiatabaci (Gennadius) biotype B: Progress report of 973 Program on invasive alien species in China［J］.Science in China Series C: Life Sciences,2009, 52: 88-95.

［21］Chen H, Chen Q, Omura T, et al.Sequential infection of rice dwarf virus in the internal organs of its insect vector after ingestion of virus［J］. Virus Research, 2011, 160(1-2): 389-394.

［22］Zhang T, Luan J B, Qi J F, et al. Begomovirus-whitefly mutualism is achieved through repression of plant defences by a virus pathogenicity factor［J］. Molecular Ecology,2012, 21(5): 1294-1304

［23］Douglas A E. Nutritional interactions in insect-microbial sysmbios,es:Aphids and their symbiotic bacteria Buchnera［J］. Annual Review of Entomology, 1998, 43: 17-37

［24］Hogenhout S A, Vander W F, Verbeek M, et al.Potato leafroll virus binds to the equatorial domain of the aphid endosymbioticGroEL homolog［J］.Journal of Virology, 1998, 72: 358-365

［25］Hogenhout S A,Vander W F, Verbeek M, et al.Indentifying the determinants in the equatorial domain of Buchnera GroEL implicated in binding potato leafroll virus［J］. Journal of Virology,2000, 74: 4541-4548.

［26］Edelbaum D, Gorovits R, Sasaki S, et al.Expressing a whitefly GroEL protein in Nicotiana benthamiana plants confers tolerance to tomato yellow leaf curl virus and cucumber mosaic virus, but not to grapevine virusaor tobacco mosaic virus［J］.Archives of Virology, 2009, 154(3): 399-407.

［27］Michael J S, Jennifer S T, Bartph J T. Plant mediated interactions between pathogenic microorganisms and herbivorous arthropods［J］. Annual Review of Entomology,2005, 51: 663-89

［28］张洁, 秦小娃, 郑雪,等.饲食感染SRBSDV的水稻病株对传毒介体白背飞虱相关能源物质含量的影响［J］.环境昆虫学报, 2013, 35(5): 597-602.(Zhang J, Qin X W, Zheng X, et al. Effects of SRBSDV－infected rice on the accumulation of energysubstances of the virus vector, Sogatellafurcifera［J］.Journal of Environmental Entomology, 2013, 35(5): 597-602.)

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备注/Memo

基金项目：国家现代农业产业技术体系建设专项(CARS-04)；公益性行业(农业)专项基金(201103002)。

第一作者简介：徐忠新(1990-)，男，硕士，主要从事大豆主要害虫综合防治研究。E-mail: zxxuneau@sina.com。

通讯作者：赵奎军（1960-），男，教授，博导，主要从事大豆主要害虫综合防治研究。E-mail: kjzhao@163.com。

更新日期/Last Update: 2016-01-05