| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2016386 | Plant Physiology and Biochemistry | 2011 | 7 Pages |
A novel cDNA encoding a cysteine proteinase inhibitor or phytocystatin was isolated from Hevea brasiliensis RRIM600 rubber latex cDNA library. The full-length HbCPI obtained from rapid amplification of cDNA ends contains 588 bp. An open reading frame of 306 bp encodes for a protein of 101 amino acids with the typical inhibitory motifs of phytocystatin superfamily, namely the central signature motif QXVXG, a GG doublet and LARFAV-like motifs in the N-terminal part, and conserved A/PW residues in the C-terminal region. Sequence comparison showed that the deduced amino acid sequence was similar to that of cysteine protease inhibitor from Manihot esculenta (84% identity). The HbCPI was subcloned into expression vector pQE-40 and then overexpressed in Escherichia coli M15 strain (pREP4) as a His-tagged recombinant protein with molecular mass approximately 13 kDa. The purified HbCPI showed thermal stable property and efficiently inhibited the protease activity of papain by non-competitive inhibition with Ki value of 15.4 nM. Beside latex, HbCPI also transcripted in leaf and young seed. The HbCPI message accumulation was induced by phytopathogenic fungi Phytophthora palmivora infection. These data suggest that HbCPI might play crucial roles in defense mechanism against biotic stimuli.
Research highlights► A new cystatin, HbCPI, was isolated from Hevea rubber latex, and its expression was found in various tissues. ► Gene organization analysis confirmed the presence of one intron located between the conserved LARFAV-like and QXVXG motifs. ► The expression of HbCPI gene in leaves could be induced upon Phytophthora palmivora infection, indicating that the gene was involved in biotic defense. ► The recombinant HbCPI also showed effective activity against papain and excellent thermal stability.
