小麦酵母双杂交文库构建及文库质量的鉴定  

Construction and Evaluation of Yeast Two-Hybrid Library of Wheat

作　　者：李立群 王冰心 吕千[1] 杨智全[1] LI Liqun;WANG Bingxin;LÜQian;YANG Zhiquan(College of Agronomy,Northwest A&F University,Yangling Shaanxi 712100,China)

机构地区：[1]西北农林科技大学农学院,陕西杨凌712100

出　　处：《西北农业学报》2022年第8期948-957,共10页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：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)。

摘　　要：以中国春小麦叶、茎、幼穗及花后3、6、9、12、15、20、25d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1.82×10^(7) CFU/mL、次级文库的容量为1.91×10^(7) CFU/mL,文库重组率均大于96%,插入片段平均长度在1000bp以上。酵母双杂交文库转化效率为1.97×10^(5)CFU/μg,文库容量为2.04×10^(7)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Ta-DAR1IP,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。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 1.82×10^(7)CFU/mL and 1.91×10^(7) CFU/mL,respectively.The inserted fragment of the cDNA library was longer than 1000 bp,and recombination rate was 96%.Transformation efficiency of yeast two-hybrid library was 1.97×10 CFU/μg,the library titer was 2.04×10^(7) 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.

关 键 词：小麦 酵母双杂交文库 自激活检测 文库筛选 

分 类 号：S512.1[农业科学—作物学]