Construction Of CDNA Library Of Alternaria And Screening For The Coding Gene Of Plant Activator Protein

A cDNA entry library and a cDNA expression library of Alternaria spp. JH505 were constructed with in vitro site-specific recombination technique, and a full-length coding sequence of plant activator protein was obtained by screening the expression library with antibody probe. At first, total RNA and mRNAs were isolated and purified from JH505 strain to synthesize double-strand cDNAs, which were ligated to attB adopter later. Through BP recombination reaction, attB-flanked cDNAs were cloned directly into an attP-containing donor vectors after size fraction to remove small size cDNAs and residual adopters, and the vectors were transformed into DH10B competent cells to generate cDNA entry library. Then a cDNA expression library was constructed through LR recombination reaction with 1×107 cfu picked from the entry library. The entry library totally contains 9×107 cfu with titer of 1×107 cfu/ml and average insert size of 1510kb, and the ratio of positive clones reaches 100%; for the expression library, the titer is 1.58×106 cfu/ml,the total number of colony-forming units 6.32×106 cfu , the average insert size 1680kb, and its ratio of positive clones also reaches 100%. In the end, through immunological screening of the expression library with the antiserum of plant activator protein, a full-length coding sequence of 1092kb that contains initiation codon and termination codon was obtained. After analysis, it was found that the protein encoded by the sequence has a molecular weight and pI very close to plant activator protein, and the sequence is preliminarily determined to be the coding sequence of plant activator protein. Through NCBI BlastN and BlastP, no sequence with identities of more than 30% was found, which means it's a new protein. The cDNA entry library and expression library provide usable materials and tools for the research of molecular biology on Alternaria, and the coding sequence screened out of the expression library makes the clone and expression of plant activator protein possible.