Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.).

Phytocystatins play multiple roles in plant growth, development and resistance to pests and other environmental stresses. A ramie ( L.) phytocystatin gene, designated as , was isolated from a ramie cDNA library and its full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence (691 bp) consisted of a 303 bp open reading frame (ORF) encoding a protein of 100 amino acids with deduced molecular mass of 11.06 kDa and a theoretical isoelectric point (pI) of 6.0. The alignment of genome DNA (accession no. MF153097) and cDNA sequences of showed that an intron (~104 bp) exists in the coding region. The BnCPI protein contains most of the highly conserved blocks including Gly⁵-Gly⁶ at the N-terminal, the reactive site motif QxVxG (QVVSG), the L-W block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (LGR FAV DDH N) block that is common among plant cystatins. BLAST analysis indicated that BnCPI is similar to cystatins from (77%), (76%), (75%) and (75%). The was subcloned into expression vector pSmart-I and then overexpressed in BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of 11.4 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA (α-benzoyl-L-arginine-2-naphthyamide), as well as the mycelium growth of some important plant pathogenic fungi. The data further contribute to our understanding of the molecular functions of BnCPI.