[Cloning of potato invertase inhibitor St-inh cDNA and its expression in E. coli and functional analysis].
A full length cDNA clone encoding invertase inhibitor was isolated by RT-PCR combined with 5' RACE from potato (S. tuberosum) tubers of cv. JH and designated as St-inh. The encoding region of St-inh is of 663bp encoding a protein of 221 amino acids. The DNA fragment including St-inh cDNA was cloned into the vector pET28a (+) and expressed successfully in E. coli. Co-incubation of the proteins produced by St-inh in E. coli and the invertase extracts from potato tubers of cv. E1, JH and tomato fruits showed that the invertase activities of potato tubers and tomato fruits decreased by 34.3%, 21% and 33.8% respectively. These results indicated that products of St-INH protein had a function of invertase inhibitors. The analysises of the nucleotide and amino acid sequences using BLAST and T-COFFEE demonstrated that St-inh cDNA was of over 95% homologous to Kunitz-type C and there was a typical domain of Kunitz-type protein [L, I, V, M]-X-D-X-[E, D, N, T, Y-[D, G]-[R, K, H, D, E, N, Q]-X-[L, I, V, M]-X(5)-Y-X-[L, I, V, M. Therefore, it was conjectured that St-inh could be a member of Kunitz-type gene family.
Cheng
Shan Han
SH
Central China Branch-Centre of Vegetable Improvement, Wuhan 430070.
Liu
Jun
J
Song
Bo Tao
BT
Xie
Cong Hua
CH
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000235:genetics / Q000378:metabolism*; D043324:beta-Fructofuranosidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D010940:Plant Proteins; D043324:beta-Fructofuranosidase