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

文献类型： 外文期刊

第一作者： Yu, Yongting

作者： Yu, Yongting;Li, Zhimin;Cheng, Yi;Gao, Chunsheng;Zeng, Liangbin;Chen, Jia;Yan, Li;Sun, Xiangping;Guo, Litao;Yan, Zhun;Yu, Yongting;Li, Zhimin;Cheng, Yi;Gao, Chunsheng;Zeng, Liangbin;Chen, Jia;Yan, Li;Sun, Xiangping;Guo, Litao;Yan, Zhun;Zhang, Gang;Zhang, Gang

作者机构：

关键词： Boehmeria nivea L.;BnCPI;cysteine protease inhibitors;intron;functional expression

期刊名称：GENES （ 影响因子：4.096； 五年影响因子：4.339 ）

ISSN： 2073-4425

年卷期： 2017 年 8 卷 10 期

页码：

收录情况： SCI

摘要： 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 (similar to 104 bp) exists in the coding region. The BnCPI protein contains most of the highly conserved blocks including Gly(5)-Gly(6) at the N-terminal, the reactive site motif QxVxG (Q(49)V(50)V(51)S(52)G(53)), the L-79-W-80 block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L(22)G(23)R(24) F(25)A(26)V(27) (DDH30)-D-28-H-29 N-31) 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 (N alpha-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.

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