堇菜科堇菜属存在金属型植物（Metallophytes)，不同生境来源的该属植物有着明显的重金属耐性差异；该属植物也富含基因编码的大环肽（cyclotides）；大环肽有金属结合（binding）作用，但强弱与序列结构有关。本项目拟：1）采用Illumina Hiseq测序、转录组比较分析和CrpExcel筛选半胱氨酸蛋白，明确华南地区20种左右的堇菜属植物重金属解毒的物质基础及其种间差异；2）采用cDNA-tag长度选择性文库（600-1200 bp)、Roche 454(+)测序和CyPerl/CyExcel（大环肽筛选工具），发掘堇菜属植物大环肽；采用ABI3730测序校对新大环肽编码序列；在分子系统层面探讨堇菜属大环肽序列金属结合潜力与进化关系；3）基于定向进化策略，构建大容量的天然的和突变的大环肽重组表达文库，采用多种抗性筛选技术，发掘解毒能力更强的天然的和人工优化的大环肽。

The Viola plants (Violaceae family) have been found with many Metallophytes, which are also rich in a kind of plant defese peptides, cyclotides. Heavy metal tolerance and metal binding abilities have been presented with distinct differences in Viola plants and cyclotides, respectively. This project intends to carry out the following works. First, Illumina Hiseq sequencing, comparative transcriptome analysis and CrpExcel (a tool for screening cysteine-rich proteins) are employed, to investigate the transcriptomes of whole wild seedlings of 20 Viola species distributed in Southern China; these works are hopeful for elucidating both material basis and interspecific differences of heavy metal detoxification in these plants. Second, four representative Viola species are selected that they are collected from heavy metals polluted soils (two species) and normal soils (two species); the comparative transcriptome analyses are further conducted between roots and shoots of these plant seedlings cultured in control and Cd-supplemented hydrophonic solutions; these works are hopeful for finding the genes associated with plant Cd tolerance. Third, tags-cDNA libraries of above Viola plant species are synthesized that they are further gel-extracted cDNA pools with 600-1200 bp in length, and Roche 454(+) sequencing, CyPerl/CyExcel and ABI3730 sequencing techniques are used for screening and validating novel cyclotide sequences predicted from the above Hiseq/454-derived transcriptome profiles of Viola species tested; sequence evolutions and metal-binding potentialities are further evaluated from our finding cyclotides and other function-known cyclotides using phylogenetical methods. In final, on the basis of the above cyclotide-exploring works, directed evolution, recombinant expression and heavy metals resistance-screening of cyclotides are conducted for harvesting native and artificial cycotides that they possesses with strong abilities of metal binding and detoxification. Overall, this project is hopeful for facilitating the theoretical and applied researches of phytoremediation based on cyclotides and Viola plants.