Abstract: The transcription factors Flowering Locus C（FLC）and SHORT VEGETATIVE PHASE
（SVP）interact to delay the flowering time in the plants of Brassica oleracea，but the molecular
mechanisms have not been thoroughly understood. In an effort to unravel the mechanisms of the protein
interactions，five FLC genes（named BoFLCy1–BoFLCy5） were cloned in Brassica oleracea. And all the
FLC family members encoded MIKC-type proteins which were classified into two groups. Group one has
two members（BoFLCy3 and BoFLCy5）with only one variational amino acid in C domain. While group two has three members（BoFLCy1，BoFLCy2 and BoFLCy4）with one or two variational amino acid positions in
K or C domain，respectively. Then yeast two-hybrid system was used in the experiments and showed that
all the BoFLCs（BoFLCy1–BoFLCy5）could act with BoSVP protein，but the three amino acids of TET
inserted into the N-terminal of BoFLCy4 may destroy the protein interaction. Furthermore ， the
β-galactosidase activities were tested and indicated that the relationships of significantly different strengths
among BoFLCs/BoSVP were BoFLCy1 > BoFLCy2 > BoFLCy3 > BoFLCy5 > BoFLCy4. The hydrophobic
amino acids，which lied in the variational positions in KC domains of BoFLCs，were helpful for the
polymerization of FLCs/SVP. And the conservative amino acids，positions of 63，77，120，121，135 and
157 in IK domains of BoFLCy4，were respectively mutated and then acted with BoSVP protein. The results
suggested that these hydrophilic or hydrophobic amino acids in K domain not in I domain regulated and
affected the interaction strength.

Key words: Brassica oleracea, flowering regulation, FLC, SVP, yeast two-hybrid system, interaction strength