黄萎病菌诱导海岛棉酵母双杂交cDNA文库构建及评价

doi:10.13560/j.cnki.biotech.bull.1985.2014.12.017

生物技术通报 ›› 2014, Vol. 0 ›› Issue (12): 105-110.doi: 10.13560/j.cnki.biotech.bull.1985.2014.12.017

黄萎病菌诱导海岛棉酵母双杂交cDNA文库构建及评价

杨君^1,2, 张艳¹ ,王伟巧¹, 荣伟¹ ,王省芬¹ ,马峙英^1,2

1. 河北农业大学农学院教育部华北作物种质资源研究与利用重点实验室河北省作物种质资源重点实验室，保定 071001; 2. 河北农业大学作物学博士后科研流动站，保定 071001

收稿日期:2014-04-28 出版日期:2014-12-08 发布日期:2014-12-12
作者简介:杨君，男，博士后，研究方向:棉花遗传育种;E-mail:yang22181@163.com;张艳为并列第一作者
基金资助:
国家自然科学基金项目(31301370)，国家自然科学基金项目(31301371)，河北省博士后科研项目(冀人社字[2013] 303 号)

Construction and Characterization of Yeast Two-Hybrid cDNA Library Derived from Roots of Gossypium barbadense Inoculated with Verticillium dahliae

^1,2Yang Jun,¹Zhang Yan,¹Wang Weiqiao,¹Rong Wei,¹Wang Xingfen,^1,2Ma Zhiying

(1. North China Key Laboratory for Crop Germplasm Resources of Education Ministry, Key Laboratory for Crop Germplasm Resources of Hebei, Department of Agriculture, Hebei Agricultural University, Baoding 071001; 2. Postdoctoral Research Station of Crop Sciences, Hebei Agricultural University, Baoding 071001)

Received:2014-04-28 Published:2014-12-08 Online:2014-12-12

摘要/Abstract

摘要： 采用 SMART^IM cDNA 合成技术，通过同源重组方法构建了海岛棉 Pima90-53 经强致病力黄萎病菌诱导的酵母双杂交 cDNA 文库。经测定，文库容量为 2.82×10⁶，滴度为 5.02×10⁸ cfu/mL。菌落 PCR 显示，重组到 pGADT7-Rec 载体上的 cDNA 片段大小集中在 500-1 500 bp 之间，重组率 93.33%。该文库完全可用于下一步互作蛋白筛选、信号转导组件确定及抗病蛋白结构和功能分析。

关键词: 黄萎病菌, 海岛棉, 酵母双杂交, cDNA文库

Abstract: A yeast two-hybrid cDNA library derived from roots of Gossypium barbadense cv. Pima90-53 inoculated with severe virulence Verticillium dahliae was constructed based on SMART^IM technique and homologous recombination reaction. Detection of the library indicated that its capacity and titer were 2.82×10⁶ and 5.02×10⁸ cfu/mL, respectively. The result from colony PCR showed that the length of insert cDNA fragments ranged from 500 to 1 500 bp on average and the recombination rate was 93.33%. These results demonstrate that the library meets the demands of the standard cDNA library, which will be useful for screening interaction proteins, determining signal transduction components and analyzing the structure and function of disease-resistant proteins in the future.

Key words: Verticillium dahliae, Gossypium barbadense, Yeast two-hybrid, cDNA library

杨君, 张艳 ,王伟巧, 荣伟 ,王省芬 ,马峙英. 黄萎病菌诱导海岛棉酵母双杂交cDNA文库构建及评价[J]. 生物技术通报, 2014, 0(12): 105-110.

Yang Jun,Zhang Yan,Wang Weiqiao,Rong Wei,Wang Xingfen,Ma Zhiying. Construction and Characterization of Yeast Two-Hybrid cDNA Library Derived from Roots of Gossypium barbadense Inoculated with Verticillium dahliae[J]. Biotechnology Bulletin, 2014, 0(12): 105-110.

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黄萎病菌诱导海岛棉酵母双杂交cDNA文库构建及评价

Construction and Characterization of Yeast Two-Hybrid cDNA Library Derived from Roots of Gossypium barbadense Inoculated with Verticillium dahliae

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