利用酵母双杂交技术筛选鉴定与番茄环纹斑点病毒NSs蛋白互作的西花蓟马蛋白

doi:10.16380/j.kcxb.2020.06.009

昆虫学报 ›› 2020, Vol. 63 ›› Issue (6): 735-743.doi: 10.16380/j.kcxb.2020.06.009

利用酵母双杂交技术筛选鉴定与番茄环纹斑点病毒NSs蛋白互作的西花蓟马蛋白

赵星月^1,2, 陈建斌³, 王曙光¹, 张晓林^1,2, 穆野^1,2, 魏辉⁴, 赵立华², 陈永对², 郑雪², 陈勇⁴, 郑立敏^3,*, 张洁^2,*

(1. 西南林业大学生命科学学院, 昆明 650224; 2. 云南省农业科学院生物技术与种质资源研究所, 云南省农业生物技术重点实验室, 农业部西南作物基因资源与种质创制重点实验室, 昆明 650205; 3. 湖南省农业科学院植物保护研究所, 长沙 410125; 4. 福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 农业部福州作物有害生物科学观测试验站, 福州 350003)

出版日期:2020-06-20 发布日期:2020-07-02

Screening and identification of the protein interacting with the NSs protein of Tomato zonate spot virus in Franiklinella ocicdentalis(Thysanoptera: Thripidae) by yeast two-hybrid technique

ZHAO Xing-Yue^1,2, CHEN Jian-Bin³, WANG Shu-Guang¹, ZHANG Xiao-Lin^1,2, MU Ye^1,2, WEI Hui⁴, ZHAO Li-Hua², CHEN Yong-Dui², ZHENG Xue², CHEN Yong⁴, ZHENG Li-Min^3,*, ZHANG Jie^2,*

(1. College of Life Sciences, Southwest Forestry University, Kunming 650224, China; 2. Yunnan Key Laboratory of Agricultural Biotechnology, Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture, Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming 650205, China; 3. Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, China; 4. Fujian Key Laboratory of Crop Pest Monitoring and Treatment, Fuzhou Crop Pest Science Observation and Experimental Station, Ministry of Agriculture, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China)

Online:2020-06-20 Published:2020-07-02

摘要/Abstract

摘要： 【目的】筛选鉴定西花蓟马Franiklinella ocicdentalis体内与番茄环纹斑点病毒(tomato zonate spot virus, TZSV)NSs蛋白互作的介体因子。【方法】利用酵母双杂交技术筛选与TZSV NSs互作的西花蓟马蛋白;进行序列分析鉴定后，将捕获的蛋白与NSs基因回转到酵母细胞，利用营养缺陷型培养基鉴定蛋白互作情况。再利用GST Pull-down技术验证TZSV NSs与鉴定出的西花蓟马蛋白的体外互作关系。【结果】构建了TZSV NSs的酵母双杂交诱饵质粒pGBKT7-NSs，确定了诱饵质粒对酵母AH109细胞无毒性，并且无自激活活性。序列分析发现与TZSV NSs互作的西花蓟马蛋白为类电压依赖性阴离子通道(voltage-dependent anion-selective channel-like, VDAC)。酵母回转实验显示TZSV NSs与西花蓟马VDAC在酵母细胞内存在特异性互作。GST Pull-down结果表明TZSV NSs与西花蓟马VDAC在体外存在相互作用。【结论】通过酵母双杂交和GST Pull-down技术，分别在酵母细胞内和体外证实了TZSV NSs和西花蓟马VDAC存在特异性互作。这些结果有助于揭示西花蓟马VDAC蛋白调控西花蓟马持久传毒的机制，为虫传病毒病的防治提供理论基础。

关键词: 西花蓟马, 酵母双杂交, 番茄环纹斑点病毒, NSs蛋白, VDAC

Abstract: 【Aim】 To screen and identify the protein interacting with the tomato zonate spot virus (TZSV) NSs protein in the western flower thrips, Franiklinella ocicdentalis. 【Methods】 The protein in F. ocicdentalis interacting with TZSV NSs was screened by yeast two-hybrid technique. After sequence analysis, the captured protein and NSs genes were cotransformed into yeast cells, and their interaction was identified by nutrient deficient medium. Furthermore, GST Pull-down was used to confirm the interaction of NSs and the identified protein in F. ocicdentalis in vitro. 【Results】 pGBKT7-NSs, a yeast two-hybrid bait plasmid of TZSV NSs, was constructed, and it showed no toxicity and no autoactivition to AH109 yeast cell. Sequence analysis showed that a voltage-dependent anion-selective channel-like (VDAC) protein of F. ocicdentalis interacted with TZSV NSs. The cotransformation test results showed that there was a specific interaction between TZSV NSs and VDAC in yeast cell. The GST Pull-down results showed that there was interaction between TZSV NSs and VDAC in vitro. 【Conclusion】 The specific interaction between TZSV NSs and F. ocicdentalis VDAC was confirmed in yeast cell and in vitro by yeast two-hybrid and GST Pull-down techniques, respectively. The results are helpful to reveal the mechanism of the protein VDAC regulating the transmission of persistent-propagative plant viruses in F. ocicdentalis and provide a theoretical basis for the prevention and control of insect-borne virus diseases.

Key words: Franiklinella ocicdentalis, yeast two-hybrid, tomato zonate spot virus, NSs protein, VDAC

赵星月, 陈建斌, 王曙光, 张晓林, 穆野, 魏辉, 赵立华, 陈永对, 郑雪, 陈勇, 郑立敏, 张洁. 利用酵母双杂交技术筛选鉴定与番茄环纹斑点病毒NSs蛋白互作的西花蓟马蛋白[J]. 昆虫学报, 2020, 63(6): 735-743.

ZHAO Xing-Yue, CHEN Jian-Bin, WANG Shu-Guang, ZHANG Xiao-Lin, MU Ye, WEI Hui, ZHAO Li-Hua, CHEN Yong-Dui, ZHENG Xue, CHEN Yong, ZHENG Li-Min, ZHANG Jie . Screening and identification of the protein interacting with the NSs protein of Tomato zonate spot virus in Franiklinella ocicdentalis(Thysanoptera: Thripidae) by yeast two-hybrid technique[J]. Acta Entomologica Sinica, 2020, 63(6): 735-743.

[1]	孙莉, 岳金金, 费东亮, 李明, 马鸣潇, 宋铭忻. 中华蜜蜂幼虫膜蛋白酵母双杂交文库的构建[J]. 昆虫学报, 2019, 62(5): 572-577.
[2]	范琳芳, 何文婧, 苏智仁, 朱家明, 罗威, 张纯, 黄立华. 斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用[J]. 昆虫学报, 2016, 59(8): 801-811.
[3]	马晓芳, 陈国庆, 张学潮, 徐海君. 柑橘木虱酵母双杂交cDNA文库的构建及评价[J]. , 2013, 56(3): 228-233.
[4]	王冰，尹姣，李克斌，曹雅忠. 酵母双杂交筛选华北大黑鳃金龟触角均一化cDNA文库[J]. , 2013, 56(10): 1127-1134.
[5]	李硕, 孙丽娟, 李醒, 熊如意, 徐秋芳, 周益军. 灰飞虱高带毒（RSV）群体酵母双杂交cDNA文库的构建[J]. , 2011, 54(11): 1324-1328.
[6]	苏宏华, 王桂荣, 郭予元. 棉铃虫成虫感觉神经元膜蛋白(SNMP)表达及其与Gqα的关系(英文)[J]. , 2010, 53(8): 864-869.
[7]	苏宏华, 王桂荣, 张永军, 梁革梅, 吴孔明, 郭予元. 利用酵母双杂交技术筛选棉铃虫触角内与G蛋白q家族α亚基（Gqα）互作的蛋白（英文）[J]. , 2010, 53(12): 1339-1344.

利用酵母双杂交技术筛选鉴定与番茄环纹斑点病毒NSs蛋白互作的西花蓟马蛋白

Screening and identification of the protein interacting with the NSs protein of Tomato zonate spot virus in Franiklinella ocicdentalis(Thysanoptera: Thripidae) by yeast two-hybrid technique

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