摘要
目的：筛选花青素合成中的关键基因查尔酮合成酶基因CHS启动子中G-box的结合蛋白,从而找到调节CHS表达的转录因子。方法：采用Matchmaker Gold Yeast One-Hybrid Library Screening System,将CHS启动子G-box序列串联后整合入酵母染色体,构建诱饵菌株;采用SMART技术合成芜菁幼苗下胚轴cDNA,将该cDNA与pGADT7-Rec表达载体共同转化诱饵菌株,通过同源重组在酵母细胞内同步进行cDNA文库的构建和筛选;用酵母菌落PCR法获得阳性克隆中的cDNA插入片段,测序后在NCBI网站进行Blast分析。结果：共筛选了2.52×106个酵母克隆,得到94个阳性克隆,菌落PCR获得了长度为0.4~2.0 kb的cDNA插入片段,并通过Blast推测了其编码蛋白。结论：实验结果证明酵母单杂交文库构建成功,初步筛选获得了G-box结合蛋白的候选蛋白,为研究CHS的表达调控奠定了基础。
Objective： In order to screen binding proteins of G-box, an important element in chalcone synthase （CHS） promoter region, and find transcriptional regulators of CHS gene. Methods： Matchmaker Gold Yeast One-Hybrid Library Screening System was employed in this study. Bait yeast strain was constructed by synthesizing oligonucleotides containing three tandem copies of G-box core sequences and integrating it into the genome of yeast. The eDNA for hypocotyls of turnip（Brassica rapa L. subsp, rapa Tsuda） was synthesized via SMART tech- nology and co-transformed into bait yeast strain with pGADT7-Rec vector, one-hybrid eDNA library was simultaneously constructed and screened directly in yeast as a result of in vivo plasmid recombination, eDNA inserts in positive clones was amplified by yeast colony PCR and analyzed through NCBI Blast after sequencing. Results： Based on the experiments, we screened 2.52×10^6 yeast clones and got 94 positive clones. Colony PCR amplification products were 0.4-2.0 kb in length and proteins encoded by them were inferred by NCBI Blast analysis. Conclusion： The results showed that the construction of yeast one-hybrid library was successful, candidate proteins for G-box binding proteins were acquired by preliminary library screening, which laid the foundation for researches on CHS expression regulation.
出处
《生物技术通讯》
CAS
2012年第4期532-536,共5页
Letters in Biotechnology
基金
国家自然科学基金(30800082
30730078)
高等学校博士学科点专项科研基金(200802251039)
中国博士后科学基金(20090460868
201003409)
东北林业大学青年拔尖人才支持计划(YTTP-1011-17)
