“Bolgogragsky”番茄cDNA文库的构建及质量评价

The Construction and Quality Evaluation of“Bolgogragsky”Tomato cDNA Library

MYB102转录因子在番茄响应低温胁迫中起着重要作用,为了进一步探究番茄转录因子MYB102的分子作用机理,筛选其蛋白,以“Bolgogragsky”为材料,从番茄的根、茎、叶、花、果以及萼片组织中提取总RNA,以其为模板合成双链cDNA,纯化后与载体pGADT7-Rec共转化入酵母Y187感受态细胞中构建酵母双杂交cDNA文库。经测定,文库容量为2.31×10^(7) CFU,cDNA文库的转化效率为7.02×10^(6) CFU/μg,文库滴度为2.5×10^(8) CFU/mL,重组率为97%,插入的cDNA片段长度主要分布在250~2000 bp。结果表明,构建的番茄cDNA文库符合酵母双杂交文库要求,可用于筛选MYB102的互作蛋白。

MYB102 transcription factor plays an important role in tomato response to low temperature stress.In order to further explore the molecular mechanism of tomato transcription factor MYB102 and screen the protein interacting with it,a tomato yeast two-hybrid cDNA library was constructed in yeast Y187 by SMART technology.The Russian tomato variety Bolgogragsky was used to construct the yeast two-hybrid cDNA library.The total RNA was extracted from tomato root,stem,leaf,flower,fruit and sepal tissue,and was used as template to synthesize double stranded cDNA.After purification,it was transformed into yeast Y187 receptive cells together with vector pGADT7-Rec to construct yeast two-hybrid cDNA library.The test showed that the library capacity was 2.31×10^(7) CFU,the cDNA library conversion efficiency was 7.02×10^(6) CFU/μg,and the library titer was 2.5×10^(8) CFU/mL.The recombination rate was 97%,and the length of the inserted double stranded cDNA fragments was mainly distributed in 250-2000 bp.The results showed that the constructed tomato cDNA library met the requirements of yeast two-hybrid library and could be used to screen MYB102 interaction proteins.