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Genes 2017, 8(10), 265; doi:10.3390/genes8100265

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

1
Institute of Bast Fiber Crops and Center for Southern Economic Crops, Chinese Academy of Agricultural Science, Changsha 410205, China
2
College of Pharmacy and Shaanxi Provincial Key Laboratory for Chinese Medicine Basis & New Drugs Research, Shaanxi University of Chinese Medicine, Xi’an 712406, China
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 30 July 2017 / Revised: 29 September 2017 / Accepted: 3 October 2017 / Published: 11 October 2017
(This article belongs to the Section Plant Genetics and Genomics)
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Abstract

Phytocystatins 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
Keywords: Boehmeria nivea L.; BnCPI; cysteine protease inhibitors; intron; functional expression Boehmeria nivea L.; BnCPI; cysteine protease inhibitors; intron; functional expression
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MDPI and ACS Style

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.

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