title: Transcriptomic screening for cyclotides and other cysteine-rich proteins in the metallophyte Viola baoshanensis.
abstract: Cysteine (Cys)-rich proteins (CRPs) are frequently associated with plant defense and stress resistance. Viola baoshanensis is a cadmium (Cd) hyper-accumulating plant whose CRPs-based defense systems are so far poorly understood. Next generation sequencing (NGS) techniques and a specialist searching tool, CrpExcel, were employed for identifying CRPs in V. baoshanensis. The transcriptome sequences of V. baoshanensis were assembled primarily from 454FLX/Hiseq2000 reads of plant cDNA sequencing libraries. CrpExcel was then used to search the ORFs and 9687 CRPs were identified, and included zinc finger (ZF) proteins, lipid transfer proteins, thaumatins and cyclotide precursors. Real-time PCR results showed that all CRP genes tested are constitutively expressed, but the genes of defensive peptides showed greater up-regulated expression than those of ZF-proteins in Cd- and/or wounding (Wd) treatments of V. baoshanensis seedlings. The NGS-derived sequences of cyclotide precursor genes were verified by RT-PCR and ABI3730 sequencing studies, and 32 novel cyclotides were identified in V. baoshanensis. In general, the metal-binding sites of ZF-containing CRPs also represented the potential vulnerable targets of toxic metals. This study provides broad insights into CRPs-based defense systems and stress-vulnerable targets in V. baoshanensis. It now brings the number of cyclotide sequences in V. baoshanensis to 53 and based on projections from this work, the number of cyclotides in the Violaceae is now conservatively estimated to be >30000.
authors.affiliation: Guangdong Pharmaceutical University, School of Biosciences and Biopharmaceutics, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangzhou 510006, China; Sun Yat-sen University, School of Life Sciences, State Key Laboratory of Biocontrol, Guangzhou 510006, China. Electronic address: zhangj80@aliyun.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Sun Yat-sen University, School of Life Sciences, State Key Laboratory of Biocontrol, Guangzhou 510006, China. Electronic address: lijtian@mail.sysu.edu.cn.
authors.forename: Jintian
authors.initials: J
authors.lastname: Li
authors.affiliation: Guangdong Pharmaceutical University, School of Biosciences and Biopharmaceutics, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangzhou 510006, China. Electronic address: zebohuang@gdpu.edu.cn.
authors.forename: Zebo
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Sun Yat-sen University, School of Life Sciences, State Key Laboratory of Biocontrol, Guangzhou 510006, China. Electronic address: andyang@tom.com.
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.affiliation: Guangdong Pharmaceutical University, School of Biosciences and Biopharmaceutics, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangzhou 510006, China. Electronic address: zhangxiaojie9205@163.com.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Guangdong Pharmaceutical University, School of Biosciences and Biopharmaceutics, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangzhou 510006, China. Electronic address: dehuali_25@163.com.
authors.forename: Dehua
authors.initials: D
authors.lastname: Li
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane 4072, QLD, Australia. Electronic address: d.craik@imb.uq.edu.au.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: The University of Melbourne, School of Botany, Parkville 3010, VIC, Australia. Electronic address: ajmb@unimelb.edu.au.
authors.forename: Alan J M
authors.initials: AJ
authors.lastname: Baker
authors.affiliation: Sun Yat-sen University, School of Life Sciences, State Key Laboratory of Biocontrol, Guangzhou 510006, China. Electronic address: shuws@mail.sysu.edu.cn.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Shu
authors.affiliation: Sun Yat-sen University, School of Life Sciences, State Key Laboratory of Biocontrol, Guangzhou 510006, China. Electronic address: lsslb@mail.sysu.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Liao
mesh_terms: D000595:Amino Acid Sequence; D047169:Cyclotides / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D059467:Transcriptome; D032413:Viola / Q000235:genetics* / Q000378:metabolism
chemical_list: D047169:Cyclotides; D010940:Plant Proteins; D003545:Cysteine
keywords: CrpExcel; Cyclotides; Cysteine-rich proteins; Transcriptome profiles; Viola baoshanensis