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

• Published in: Genes 2017-10, Vol.8 (10), p.265
• Main Authors: Yu, Yongting, Zhang, Gang, Li, Zhimin, Cheng, Yi, Gao, Chunsheng, Zeng, Liangbin, Chen, Jia, Yan, Li, Sun, Xiangping, Guo, Litao, Yan, Zhun
• Format: Article
• Language: English
• Subjects: Antifungal activity; Arginine; Boehmeria nivea; Cystatins; Deoxyribonucleic acid; DNA; Fungi; Gel electrophoresis; Genomes; Nucleotide sequence; Papain; Pests; Proteins; Sodium lauryl sulfate
• ISSN: 2073-4425; 2073-4425
• DOI: 10.3390/genes8100265

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 (Q V V S G ), the L -W block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L G R F A V D D H 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.