[1]周晨,韩召军.灰飞虱水通道蛋白基因克隆及其与杀虫剂代谢的关系[J].南京农业大学学报,2017,40(6):1014-1022.[doi:10.7685/jnau.201703003]
 ZHOU Chen,HAN Zhaojun.Aquaporin gene cloning and its possible contribution to insecticide metabolism in Laodelphax striatellus[J].Journal of Nanjing Agricultural University,2017,40(6):1014-1022.[doi:10.7685/jnau.201703003]
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灰飞虱水通道蛋白基因克隆及其与杀虫剂代谢的关系()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
40卷
期数:
2017年6期
页码:
1014-1022
栏目:
出版日期:
2017-11-10

文章信息/Info

Title:
Aquaporin gene cloning and its possible contribution to insecticide metabolism in Laodelphax striatellus
作者:
周晨 韩召军
南京农业大学植物保护学院/农作物生物灾害综合治理教育部重点实验室, 江苏 南京 210095
Author(s):
ZHOU Chen HAN Zhaojun
College of Plant Protection/Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China
关键词:
灰飞虱水通道蛋白基因克隆体外表达杀虫剂转运
Keywords:
Laodelphax striatellusaquaporingene cloningfunctional expressioninsecticide transport
分类号:
S435.112+.3
DOI:
10.7685/jnau.201703003
摘要:
[目的] 水通道蛋白是细胞膜内嵌蛋白超家族的主要成员,具有传输水分子以及小分子化合物进出细胞膜的功能,本研究目的是分析重要农业害虫灰飞虱体内水通道蛋白功能及其与杀虫剂代谢之间的关系。[方法] 根据转录组数据,采用PCR和RACE技术对灰飞虱体内的水通道蛋白基因进行了克隆,并利用进化树对获得的基因进行亲缘关系的分析验证,利用实时荧光定量PCR技术分析这些基因在灰飞虱抗、感品系间的表达量差异,利用体外表达技术分析其转运活性。[结果] 从灰飞虱体内克隆获得了6个灰飞虱水通道蛋白基因全长序列,分别命名为LsAQP1、LsAQP2、LsAQP3、LsAQP4、LsAQP5LsAQP6,其中LsAQP6在灰飞虱的不同抗性品系里均高表达。体外功能表达试验显示:LsAQP6对水具有很强的转运能力,对灭多威也具有显著的转运能力,但对甘油和杀虫双的转运能力不显著。[结论] LsAQP6在灰飞虱体内对水和某些小分子农药具有转运作用。
Abstract:
[Objectives] Aquaporin, a member of the major intrinsic protein superfamily, functions mainly for transporting water and small molecule compounds across cell membrane, and the aim of this study was to analyze the function of the aquaporin and its possible contribution to insecticide metabolism in an important agricultural pest Laodelphax striatellus.[Methods] In this study, aquaporin genes in L. striatellus were cloned based on the transcriptome data and with PCR and RACE cloning techniques. The cloned genes were subjected to phylogenetic relationship analysis by constructing phylogenetic tree with previously reported insect aquaporin genes, tests of the expression level in the susceptible and resistant strains by real-time quantitative PCR, and functional verification for their transporting capability by combinant expression in vitro.[Results] Six full length aquaporin genes were cloned from L. striatellus and designated as LsAQP1, LsAQP2, LsAQP3, LsAQP4, LsAQP5 and LsAQP6, respectively. Among them, LsAQP6 was found expressed with higher level in all three resistant strains tested as compared to the susceptible strain. Furthermore, functional expression showed that LsAQP6 had strong transport capacity for water and significant transport capacity for methomyl, but not for glycerol and bisultap.[Conclusions] Thus, it is speculated that LsAQP6 plays a significant role in transporting water and some small molecule insecticides in L. striatellus.

参考文献/References:

[1] Campbell E M,Ball A,Hoppler S,et al. Invertebrate aquaporins:a review[J]. J Comp Physiol B,2008,178(8):935-955.
[2] 刘海远,舒本水,姜春来,等. 斜纹夜蛾水通道蛋白1(AQP1)基因的克隆、分子特性和表达分析[J]. 昆虫学报,2013,56(4):339-349. Liu H Y,Shu B S,Jiang C L,et al. Molecular cloning,characterization and expression analysis of aquaporin 1(AQP1)gene in Spodoptera litura(Lepidoptera:Noctuidae)[J]. Acta Entomologica Sinica,2013,56(4):339-349(in Chinese with English abstract).
[3] Song J,Mak E,Wu B,et al. Parasite aquaporins:current developments in drug facilitation and resistance[J]. Biochim Biophys Acta,2014,1840(5):1566-1573.
[4] Graf F E,Ludin P,Wenzler T,et al. Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol[J]. PLoS Negl Trop Dis,2013,7(10):e2475.
[5] 胡兴强. 家蝇抗药性机理研究综述[J]. 安徽预防医学杂志,2003,9(5):336-339. Hu X Q. A review of studies on resistance mechanism of musca domestica[J]. Anhui Journal of Preventive Medicine,2003,9(5):336-339(in Chinese).
[6] 胡小邦,朱昌亮. 昆虫抗药性机制研究进展[J]. 国外医学:寄生虫病分册,2004,31(1):18-23. Hu X B,Zhu C L. Advances in insect resistance mechanism[J]. Foreign Med Sci:Parasit Dis,2004,31(1):18-23(in Chinese).
[7] 曲明静,许新军,韩召军,等. 昆虫乙酰胆碱酯酶基因变异抗药性机制研究[J]. 昆虫知识,2007,44(2):191-194. Qu M J,Xu X J,Han Z J,et al. Advances in studies of acetylcholinesterase gene variation assoiated with insect resistance[J]. Chinese Bulletin of Entomology,2007,44(2):191-194(in Chinese with English abstract).
[8] 李晨歌,琚阳,韩召军,等. 马铃薯甲虫3个烟碱型乙酰胆碱受体α亚基基因的克隆及表达分析[J]. 南京农业大学学报,2014,37(3):65-71. DOI:10.7685/j.issn.1000-2030.2014.03.009. Li C G,Ju Y,Han Z J,et al. Cloning and expression analysis of three genes encoding nicotinic acetylcholine receptor α subunit in Leptinotarsa decemlineata[J]. Journal of Nanjing Agricultural University,2014,37(3):65-71(in Chinese with English abstract).
[9] 王芮,符蓉,杨倩文,等. 抗药性灰飞虱稳定内参基因的筛选[J]. 南京农业大学学报,2016,39(5):763-770. DOI:10.7685/jnau.201511031. Wang R,Fu R,Yang Q W,et al. Identification of reference genes in resistant small brown plant hopper,Laodelphax striatellus[J]. Journal of Nanjing Agricultural University,2016,39(5):763-770(in Chinese with English abstract).
[10] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[11] 李仁. 番茄水通道蛋白在干旱胁迫及果实成熟过程中的功能分析[D]. 北京:中国农业大学,2015. Li R. Characterization of aquaporin genes in tomato during drought stress and fruit development process[D]. Beijing:China Agricultural University,2015(in Chinese with English abstract).
[12] Wallace I S,Roberts D M. Distinct transport selectivity of two structural subclasses of the nodulin-like intrinsic protein family of plant aquaglyceroporin channels[J]. Biochemistry,2005,44:16826-16834.
[13] Wallace I S,Shakesby A J,Hwang J H,et al. Acyrthosiphon pisum AQP2:a multifunctional insect aquaglyceroporin[J]. Biochim Biophys Acta,2012,1818(3):627-635.
[14] Staniscuaski F,Paluzzi J P,Real-Guerra R,et al. Expression analysis and molecular characterization of aquaporins in Rhodnius prolixus[J]. J Insect Physiol,2013,59(11):1140-1150.
[15] van Ekert E,Chauvigné F,Finn R N,et al. Molecular and functional characterization of Bemisia tabaci aquaporins reveals the water channel diversity of hemipteran insects[J]. Insect Biochem Mol Biol,2016,77:39-51.
[16] 王勋. 水稻二化螟CsAQP1基因的克隆与表达[D]. 扬州:扬州大学,2011. Wang X. Cloning and experession of CSAQP1 genes in rice stem borer,Chilo suppressalis Walker(Lepidoptera:Pyralidae)[D]. Yangzhou:Yangzhou University,2011(in Chinese with English abstract).
[17] Verkman A S. Aquaporins in clinical medicine[J]. Annu Rev Med,2012,63:303-316.
[18] Verkman A S,Anderson M O,Papadopoulos M C. Aquaporins:important but elusive drug targets[J]. Nat Rev Drug Discov,2014,13(4):259-277.
[19] Li G,Santoni V,Maurel C. Plant aquaporins:roles in plant physiology[J]. Biochim Biophys Acta,2014,1840(5):1574-1582.
[20] Tornroth-Horsefield S,Hedfalk K,Fischer G,et al. Structural insights into eukaryotic aquaporin regulation[J]. FEBS Lett,2010,584(12):2580-2588.
[21] Agre P,King L S,Yasui M,et al. Aquaporin water channels-from atomic structure to clinical medicine[J]. The Journal of Physiology,2002,542(1):3-16.
[22] Benoit J B,Hansen I A,Attardo G M,et al. Aquaporins are critical for provision of water during lactation and intrauterine progeny hydration to maintain tsetse fly reproductive success[J]. PLoS Negl Trop Dis,2014,8(4):e2517.
[23] Drake L L,Boudko D Y,Marinotti O,et al. The aquaporin gene family of the yellow fever mosquito,Aedes aegypti[J]. PLoS ONE,2010,5(12):e15578.
[24] Liu K,Tsujimoto H,Cha S J,et al. Aquaporin water channel AgAQP1 in the malaria vector mosquito Anopheles gambiae during blood feeding and humidity adaptation[J]. Proc Natl Acad Sci USA,2011,108(15):6062-6066.
[25] Pietrantonio P V,Jagge C,Keeley L L,et al. Cloning of an aquaporin-like cDNA and in situ hybridization in adults of the mosquito Aedes aegypti(Diptera:Culicidae)[J]. Insect Mol Biol,2000,9(4):407-418.
[26] Duchesne L,Hubert J F,Verbavatz J M,et al. Mosquito(Aedes aegypti)aquaporin,present in tracheolar cells,transports water,not glycerol,and forms orthogonal arrays in Xenopus oocyte membranes[J]. Eur J Biochem,2003,270(3):422-429.
[27] Cohen E. Roles of aquaporins in osmoregulation,desiccation and cold hardiness in insects[J]. Entomology,Ornithology and Herpetology,2012,S1:001.
[28] Drake L L,Rodriguez S D,Hansen I A. Functional characterization of aquaporins and aquaglyceroporins of the yellow fever mosquito,Aedes aegypti[J]. Sci Rep,2015,5:7795.
[29] Baker N,Glover L,Munday J C,et al. Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes[J]. Proc Natl Acad Sci USA,2012,109(27):10996-11001.
[30] Li R,Wang J,Li S,et al. Plasma membrane intrinsic proteins SlPIP2;1,SlPIP2;7 and SlPIP2;5 conferring enhanced drought stress tolerance in tomato[J]. Sci Rep,2016,6:31814.

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备注/Memo

备注/Memo:
收稿日期:2017-03-02。
基金项目:国家自然科学基金项目(31130175)
作者简介:周晨,硕士研究生。
通信作者:韩召军,教授,博导,研究方向为昆虫生理生化与分子生物学,E-mail:zjhan@njau.edu.cn。
更新日期/Last Update: 1900-01-01