Cloning and knockout vector construction of SlCmr1 gene in Stemphylium lycopersici

【Objective】In order to study the function of SlCmr1 gene in Stemphylium lycopersici, cloning of SlCmr1 gene was carried out, and gene knockout vector was constructed.【Method】The primers of SlCmr1 gene were designed according to the S. lycopersici genome sequence provided by NCBI database. The full length DNA and CDS sequence of SlCmr1 gene were obtained by PCR amplification and the protein coded by SlCmr1 gene was analyzed through bioin formatics methods. Sequences around 1100 bp upstream and downstream of SlCmr1 gene were used to design primers. The upstream and downstream sequences of SlCmr1 were amplified by PCR and inserted into pCX62 vector by restriction enzyme digestion and SlCmr1 gene knockout vector was constructed.【Result】SlCmr1 gene was 3275 bp in DNA sequence and 3018 bp in CDS sequence, including 3 introns, encoded 1005 amino acid protein sequence with molecular formula C₄₈₈₂H₇₆₄₂N₁₄₀₄O₁₄₉₉S₆₁ and molecular weight 111.94 kD, predicted isoelectric point(pI) 6.64, halflife 30 h, instability index 58.72, mean value of hydrophilicity/hydrophobicity-0.424 and subcellular localization in nucleus. It was speculated that SlCMR1 was a non-secretory, unstable soluble protein without transmembrane region, and there were 2 adjacent ZnF_C2H2 domains and 1 GAL4 domain. SlCmr1 gene knockout vector pCX62-SlCmr1 was successfully constructed by inserting the upstream and downstream fragments of SlCmr1 gene into pCX62 vector.【Conclusion】Slcmr1 may be a key gene regulating pigment synthesis and play an important role in the secondary metabolite synthesis in fungi.