›› 2011, Vol. 54 ›› Issue (11): 1324-1328.doi:

• 简报 • 上一篇    下一篇

灰飞虱高带毒(RSV)群体酵母双杂交cDNA文库的构建

 李硕, 孙丽娟, 李醒, 熊如意, 徐秋芳, 周益军   

  • 收稿日期:2011-07-29 修回日期:2011-09-25 出版日期:2011-11-20 发布日期:2011-11-20
  • 通讯作者: 周益军 E-mail:yjzhou@jaas.ac.cn
  • 作者简介:李硕, 男, 1984年5月生, 山东淄博人, 博士研究生, 主要从事植物病毒与昆虫介体互作研究, E-mail: lishuolstonnv989@163.com; 孙丽娟, 女, 1985年6月生, 江苏盐城人, 硕士研究生, 主要从事植物病毒学研究, E-mail: 2009102016@njau.edu.cn
  • 基金资助:

    基金项目: 国家重点基础研究发展规划(“973”计划)项目(2010CB126203); 国家自然科学基金项目(30970132); 国家公益性行业(农业)科研专项(201003031); 江苏省自然科学基金项目(BK2010018)

Construction of yeast two-hybrid cDNA library of high-viruliferous (RSV-infected) populations of the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae)

 LI  Shuo, SUN  Li-Juan, LI  Xing, XIONG  Ru-Yi, XU  Qiu-Fang, ZHOU  Yi-Jun   

  • Received:2011-07-29 Revised:2011-09-25 Online:2011-11-20 Published:2011-11-20
  • Contact: ZHOU Yi-Jun E-mail:yjzhou@jaas.ac.cn
  • About author:lishuolstonnv989@163.com;2009102016@njau.edu.cn

摘要: 为了研究灰飞虱Laodelphax striatellus Fallén与水稻条纹病毒(rice stripe virus, RSV)互作机制, 本研究构建了灰飞虱高带毒群体酵母双杂交cDNA文库。以实验室筛选的灰飞虱高带毒群体为材料, 分离纯化mRNA, 反转录合成双链cDNA, 并连接三框型接头, 层析柱分级纯化。采用同源重组反应制备三框型cDNA入门文库, 再通过同源重组将入门文库转移到Gateway兼容载体pGADT7-DEST上, 构建获得酵母双杂交cDNA文库。检测结果表明: 文库库容量为3.68×107 cfu, 扩增文库滴度为2.62×1010 cfu/mL; 文库重组率大于95%, cDNA插入片段平均长度>1 kb, 达到了标准cDNA文库的要求。灰飞虱高带毒群体酵母双杂交cDNA文库的构建为开展昆虫介体与水稻条纹病毒互作机制的研究奠定了基础。

关键词: 灰飞虱, 水稻条纹病毒, 同源重组, cDNA文库, 酵母双杂交cDNA文库

Abstract:  In order to research the interaction between the small brown planthopper (Laodelphax striatellus Fallén, SBPH) and rice stripe virus (RSV), yeast two-hybrid cDNA library of high-viruliferous (RSV-infected) SBPH populations was constructed. SBPH infected with RSV was maintained in our laboratory. The mRNA was isolated and purified from high-viruliferous populations, and used as the template to synthesize the double-strand cDNAs. The cDNAs were ligated with three-frame adapter and purified by the cDNA size fractionation columns. By using homologous recombination reaction, the cDNA entry library was prepared and then recombined into Gateway compatible vector pGADT7-DEST to construct yeast two-hybrid cDNA library. Detection of the library indicated that it contained 3.68×107 independent clones, and the titer of the amplified library was 2.62×1010 cfu/mL. The recombination rate was above 95%, and the average size of inserts was above 1 kb in the cDNA library. The results demonstrate that the library database meets the requirements of the standard cDNA library. The yeast two-hybrid cDNA library of high-viruliferous SBPH will be useful for the future research on the interaction between insect vector and RSV.

Key words: Small brown planthopper (Laodelphax striatellus), rice stripe virus, homologous recombination, cDNA library, yeast two-hybrid cDNA library