Cloning and Functional Analysis of the MADS-box CiMADS9 Gene from Carya illinoinensis

A MADS-box gene, CiMADS9, was cloned from the male flowers of Carya illinoinensis by rapid amplification of cDNA ends. The gene was 1 077bp with a 768bp open reading frame encoding 255 amino acids. Multiple sequence comparisons revealed that CiMADS9 is a typical MIKC-type MADS-box gene with a MADS-box domain and a K semi-conserved region. Phylogenetic analysis indicated that CiMADS9 belongs to the AGL15 group of the MADS-box gene family. Quantitative reverse transcription polymerase chain reaction analysis indicated that the expression levels in reproductive organs (i.e., flowers and young fruits) were considerably higher than in vegetative tissues (i.e., leaves and branches). The highest expression levels were observed in male flowers. An overexpression vector for CiMADS9 was constructed and the gene was inserted into the Arabidopsis thaliana genome. CiMADS9 expression was confirmed in all transgenic lines. Compared with wild-type plants, transgenic A. thaliana plants overexpressing CiMADS9 exhibited delayed flowering and an increased number of leaves.