Cloning and Analyzing of AP3-3 and Its Promoter from Dendrobium officinale

  Objective  AP3-3 of class B gene of MADS box family that involves in the formation of perianth and labellum of Dendrobium officinale was cloned to study the biological function, and the promoter analyzed to decipher the regulation mechanism.

  Method  Sequences of AP3-3 and promoter of D. officinale were cloned by RT-PCR and conventional PCR, and bioinformatics analyzed. A fusion expression vector of promoter-deleted fragments and GUS gene was constructed. Agrobacterium tumefaciens-mediated transformation was used to transform protocorm of D. officinale for the transient expression.

  Result  The cDNA length of DoAP3-3 was 675 bp with an encoded formula of C₁₁₂₉H₁₈₀₃N₃₃₃O₃₄₇S₁₂, a molecular weight of 25.98 kDa, a PI of 8.71, an instability index of 40.14, and GRAVY of −0.823. There was no transmembrane region detected in the protein. The predicted score of subcellular localization was 87.0% in nucleus, 8.7% in mitochondria, and 4.3% in cytoplasm. The 1885 bp promoter fragment had a cis acting element containing a significant number of photo-responsive elements among others. The 3 promoter fragments could drive GUS with an order of expression intensity of −885–0 bp＞−1 604–0 bp＞−750–0 bp.

  Conclusion  The predicted DoAP3-3 was an alkali, hydrophilic, and unstable protein with no transmembrane domain and a subcellular localization in the nucleus. The DoAP3-3 promoter might be regulated by light, plant hormones, MYB transcription protein, etc., and exhibited activities that increased with decreasing deletion length.