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小麦酵母双杂交文库构建及文库质量的鉴定
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引用本文:李立群,王冰心,吕 千,杨智全.小麦酵母双杂交文库构建及文库质量的鉴定[J].西北农业学报,2022,(8):948~947
DOI:10.7606/j.issn.1004-1389.2022.08.002
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作者单位
李立群,王冰心,吕 千,杨智全 (西北农林科技大学 农学院,陕西杨凌 712100) 
基金项目:陕西省农业协同创新与推广联盟重大科技项目(LMZD202104);西北农林科技大学实验技术研究与实验室管理创新项目(SY20210227)。
中文摘要:以中国春小麦叶、茎、幼穗及花后3、6、9、12、15、20、25 d的籽粒为材料,利用Gateway技术构建中国春小麦cDNA初级文库和次级文库,由次级文库质粒转化Y187酵母菌株构建出酵母双杂交文库。通过mating法筛选小麦粒质量基因TaDAR1的互作蛋白,以此来鉴定文库的有效性。结果表明:初级文库容量为1.82×107 CFU/mL、次级文库的容量为1.91×107 CFU/mL,文库重组率均大于 96%,插入片段平均长度在 1 000 bp 以上。酵母双杂交文库转化效率为1.97×10 CFU/μg,文库容量为2.04×107 CFU/mL。构建诱饵载体pGBKT7-TaDAR1并转化Y2H酵母菌株,经检验存在自激活现象,进一步截断检测发现此蛋白N端无自激活,C端有自激活,以BD-TaDAR1-N为诱饵载体,筛选酵母文库,获得其互作蛋白序列,并命名为TaDAR1IP,经回转验证TaDAR1-N与TaDAR1IP存在相互作用关系。表明本试验方案构建的酵母文库是高质量的有效的。为初学者构建酵母双杂文库及使用“mating”法筛选酵母文库提供借鉴,也为后续小麦基因功能的研究奠定基础。
中文关键词:小麦  酵母双杂交文库  自激活检测  文库筛选
 
Construction and Evaluation of Yeast Two-Hybrid Library of Wheat
Abstract:Yeast two-hybrid(Y2H) library is the base of gene screening,identification of the interaction proteins and elucidation of the mechanism. In this study, the leaves, stems, young spirkes and the grains at 3,6,9,12,15,20,25 d of the ‘Chinese spring ’ wheat after flowering were used as materials to construct the primary and secondary cDNA libraries by gateway technique. Finally, Y2H library was established by transforming secondary library plasmid to Y187. Two-hybrid library screening was performed by mating following detection of the self-activating and toxicity of the bait vector pGBKT7-TaDAR1 in yeast. The results showed that the library titer of primary and secondary cDNA libraries were 1.82×107CFU/mL and 1.91×107 CFU/mL, respectively. The inserted fragment of the cDNA library was longer than 1 000 bp, and recombination rate was 96%. Transformation efficiency of yeast two-hybrid library was 1.97×10 CFU/μg, the library titer was 2.04×107 CFU/mL. We found that the bait vector of BD-TaDAR1 had self-activation effect,but the BD-TaDAR1-N did not. So the bait vector with BD-TaDAR1-N was used to screen Y2H library. One potential interacting protein TaDAR1IP was obtained. The results indicated that the library was of high quality and effective. This study lays a foundation for subsequent function verification of wheat functional genes, and provides some experimental guidance for beginners to understand the construction and application of yeast two-hybrid cDNA library as well.
keywords:Wheat  Yeast two-hybrid cDNA Library  Self-activation detection  Library screening
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