Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.)
AbstractPhytocystatins play multiple roles in plant growth, development and resistance to pests and other environmental stresses. A ramie (Boehmeria nivea L.) phytocystatin gene, designated as BnCPI, 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 BnCPI 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 (Q49V50V51S52G53), the L79-W80 block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L22G23R24 F25A26V27 D28D29H30 N31) block that is common among plant cystatins. BLAST analysis indicated that BnCPI is similar to cystatins from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%). The BnCPI was subcloned into expression vector pSmart-I and then overexpressed in Escherichia coli 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 (Nα-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. View Full-Text
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Yu, Y.; Zhang, G.; Li, Z.; Cheng, Y.; Gao, C.; Zeng, L.; Chen, J.; Yan, L.; Sun, X.; Guo, L.; Yan, Z. Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.). Genes 2017, 8, 265.
Yu Y, Zhang G, Li Z, Cheng Y, Gao C, Zeng L, Chen J, Yan L, Sun X, Guo L, Yan Z. Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.). Genes. 2017; 8(10):265.Chicago/Turabian Style
Yu, Yongting; Zhang, Gang; Li, Zhimin; Cheng, Yi; Gao, Chunsheng; Zeng, Liangbin; Chen, Jia; Yan, Li; Sun, Xiangping; Guo, Litao; Yan, Zhun. 2017. "Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.)." Genes 8, no. 10: 265.
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