西花蓟马酵母双杂交文库及TSWV 膜蛋白诱饵载体的构建
Construction of a western flower thrip yeast two-hybrid library and TSWV membrane protein bait vectors
万岩然1** 郑晓斌2 袁江江2 张友军2 吴青君2***
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DOI：10.7679/j.issn.2095-1353.2019.012
作者单位：1. 河北农业大学植物保护学院，保定 071000；2. 中国农业科学院蔬菜花卉研究所，北京 100081
中文关键词：西花蓟马；番茄斑萎病毒；酵母双杂交文库；诱饵载体
英文关键词： Frankliniella occidentalis; tomato spotted wilt virus; yeast two hybrid library; bait vector
中文摘要：
【目的】 为筛选西花蓟马Frankliniella occidentalis与番茄斑萎病毒的互作蛋白。【方法】 利用Make Your Own“Mate & Plate™”Library System构建西花蓟马酵母双杂交初级cDNA文库以及Y187酵母次级文库，利用Matchmaker Gold Yeast Two-Hybrid System构建TSWV膜蛋白GN及GC诱饵载体。【结果】 建立的西花蓟马初级cDNA文库库容为5.6×106 cfu，其插入片段平均长度大于1 000 bp，重组率约为100%。建立的Y187酵母次级文库转化效率为5×106 cfu/μg，文库滴度为2.97×108。构建的pGNKT7-GN及pGBKT7-GC诱饵载体能够在Y2HGold酵母菌株中正确表达，无自激活活性且无毒性。【结论】 成功构建了西花蓟马酵母双杂交文库以及番茄斑萎病毒膜蛋白诱饵载体，可用于后续筛库实验。
英文摘要：
[Objectives] To screen proteins involved
in the interaction between western flower thrips and the Tomato spotted wilt virus (TSWV). [Methods] A western flower thrip cDNA library and a
Y187 yeast library were constructed using the Make Your Own “Mate & Plate™”
Library System, and TSWV GN and GC bait vectors were constructed using the
Matchmaker Gold Yeast Two-Hybrid System. [Results] The capacity of the western flower thrip cDNA
library was 5.6×106 cfu，the average library recombination
rate was about 100%, and the average amplification sizes of insert fragments in
the cDNA library were above 1 kb. The transformation ratio and titer of the
Y187 yeast library were 5×106 cfu/μg and 2.97×108, respectively. The
constructed pGNKT7-GN and pGBKT7-GC bait vectors were well expressed in yeast
and had no self-activation and toxicity. [Conclusion] A western flower thrip yeast library and TSWV
membrane protein bait vectors were successfully constructed and can be used for
further hybrid library screening.