Position: Home > Articles > Construction of a Yeast-Two-Hybrid Library of Chlamydomonas reinhardtii with SMART Technique
Biotechnology Bulletin
2011,0
(9)
187-192
SMART技术构建衣藻细胞的酵母双杂交文库
作 者：
孙银行;潘俊敏
单 位：
清华大学生命科学学院细胞骨架与信号转导实验室
关键词：
鞭毛再生;细胞周期;衣藻;酵母双杂交文库;SMART技术
摘 要：
衣藻是用来研究植物光合作用和动物纤毛常用的模式生物。为了研究蛋白质之间的相互作用,利用SMART技术构建了衣藻的酵母双杂交文库。使用Trizol试剂提取鞭毛再生过程和光周期培养的细胞进入分裂前的衣藻细胞的总RNA,经过Oligotex纯化得到mRNA;应用SMART技术和LD-PCR合成双链cDNA,经过CHROMA SPIN TE-400柱子去除短片段的cDNA;cDNA和线性化载体pGADT7-Rec共转化酵母Y187构建酵母双杂交文库。库容达到3.0×106 CFU,重组率为70%,插入片段平均长度为0.6 kb。以上结果说明该文库质量较好,能够通过筛选文库得到与目的蛋白互相作用的蛋白质,为寻找蛋白质的作用伴侣打下基础。
译 名：
Construction of a Yeast-Two-Hybrid Library of Chlamydomonas reinhardtii with SMART Technique
作 者：
Sun Yinhang Pan Junmin (Cytoskeleton and Signal Transduction Lab,School of Life Science,Tsinghua University,Beijing 100084)
关键词：
Flagella regeneration Cell division Chlamydomonas reinhardtii Yeast-two-hybrid library SMART technique
摘 要：
A Yeast-Two-Hybrid library of the model organism Chlamydomonas reinhardtii was constructed.Total RNA was extracted from cells undergoing flagellar assembly or prior to mitosis using Trizol reagent.Oligotex was used to purify mRNA.The first strand cDNA was synthesized by reverse transcription of mRNA with SMART technique,and LD-PCR was performed to synthesize double strand cDNA.Shorter cDNAs were eliminated by running through a CHROMA SPIN TE-400 column.Purified cDNA and linear vector(pGADT7-Rec)co-transformed into yeast strain,Y187,generating Yeast-Two-Hybrid library.The capacity of the library is 3.0×106 CFU with a recombination rate of 70% and an average size of inserts of 0.6 kb.These results indicate that the library is suitable for screening for potential partner(s)of proteins of interest.
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