Construction Of Pepper CDNA Library With Leaves Challenged By UV And The Library Screening For WRKY's CDNA

Transcription factors, such as WRKY, play important roles in the regulation of plant tolerance to environmental stresses, to isolate such kind of importmant transcription factors for further functional study, an cDNA library was constructed with the UV treated Capsicum annuum leaves on the basis of total RNA extraction method optimization and an positive cDNA clone of WRKY was isolated from the cDNA library. The main results were as following:1. Construction of cDNA libraryTotal RNA extraction method was optimized with the pepper leaves treated with UV radiation, and the total RNA extracted by the optimized method could meet the requirement of cDNA library construction. The cDNA library was contructed with SMART? cDNA library construction kit according to the enclosed User Manual, the first strand cDNA was synthesized from the total RNA with the one point mutant of Moloney murine leukemia virus (MMLV) reverse transcriptase, and the dscDNA was Amplified by LD PCR. After size-fraction by CHROMA SPIN-400 Column to remove the fraction shorter than 500bp, the dscDNA was further cloned intoλTriplEx2 vector, the resulting ligating material was λ-phage packaged and the unamplified cDNA library with a titer more than 2.17×l0~6pfu was achieved. The unamplified cDNA library was further amplified and the titer of the amplified cDNA was 3.77-3.89×10~8pfu/ml, the recombinant efficiency was 93% by randomly PCR determination, the cDNA library could be used for cDNA cloning.2. Isolation of WRKY by PCR based 96-hole library screening method.EST of Capsicum annuum was blasted from the GenBank using the amino acid sequence of WRKY of Arabidopsis thaliana as probe, and the ESTs were further assemblied by DNAMAN, and several pairs of specific primers were designed and synthesized according to the homology region of the assembly tentative WRKY sequences. With the above WRKY specific primers, a positive clone was isolated by...