Cloning And Sequence Analysis Of The Gene Encoding Voltage-Gated Chloride Channel In Vicia Faba Leaves

The discovery of the ion channel in the Vicia faba guard cell has gained of great impetus of development in the membrane level of K⁺, Ca2⁺,Cl^- and malate channel system, ranging from characteristic traits, physiological functioning, to the molecular mechanism aspects. Chloride Channel （CLCs）, one of the most important anion channels, is serving more important role in the procedure of cell turgor, osmoregulation, ionic homeostasis, pH environment, stomatal movement, nutrients transduction, mental tolerance and signal identification and transduction.In this study, we analyzed the homogenous of CLC gene in different species and tried to target the conservative region of amino acids by gene sequence comparison. The 824bp cDNA sequence cloned by using RT-PCRwith the Primer 5.0 derivative primer, was identified as CLC family with BLAST analysi. Then the 3’RACE primer was designed to clone a 1587bp CLCs cDNA sequence which contained an open reading frame, 901bp, 32445.3Da, and the molecular formula is inferred C₁478H₂361N₃73O₄07S₁8. This sequence contained 20 negative charged （Asp+Glu） and 20 positive charged（Arg+Lys） amino acid respectively, and isoelectric point of the protein 7.19 therotical. Naming the CLC as VfCLC（Vicia faba voltage-gated chloride channel）. The homology analysis suggest that the protein belongs to CLC family, and it is highest related to GmCLC, 87.3%.Hydrophobicity analysis showed that this N-terminal is hydrophobicity, and C-terminal is hydrophily. The maxim and minima of hydrophobicity is 3.156, -2.189 respectively. Further analysis indicated, a signal peptide dissection site between the 31th and the 32th amino acid. The prediction of transmembrane region showed four transmembrane helix, which indicated a transmembrane protein type.