华南堇菜属植物的比较转录组分析与重金属解毒大环肽定向进化

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.

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。

华南堇菜属植物有20余种，多分布于岭南山地正常土壤中，部分种见于金属矿山；有着3对二硫键、首尾环化、长28-37 aa大环肽（Cyclotides）在堇菜属植物中的分布极为典型。本项目工作在以下几个方面取得进展。（1）采集区域内20种堇菜属植物，进行二代RNA测序，组装转录组，筛选近200个环肽序列；（2）对宝山堇菜进行深度基因组测序，选择5种堇菜开展基因组重测序研究，筛选近500个环肽；（3）选择宝山堇菜和长萼堇菜开展比较转录组水平研究，宝山堇菜体有更为活跃的泛素化调控蛋白质水解过程和液泡膜转运蛋白，糖代谢可能通过调控细胞壁修饰响应重金属胁迫。（4）基于分子系统进化关系，选择代表性环肽开展重组表达与功能研究，并对部分环肽进行定向突变的结构和功能研究，结果鉴定大环肽viphi I有多种植物防卫活性和对重金属Cd有较强的束缚作用；（5）开展大环肽在细胞筛选和防天然抗生素抗性菌污染的线虫模型研究。从组学层面证实大环肽在华南堇菜属植物中分布极为丰富，大环肽有着重要的植物防卫功能，对金属型植物广泛存在的堇菜属植物的重金属稳定有着重要作用。项目工作发表了7篇学术论文，3篇SCI论文，影响因子均大于4。在人才培养方面，本项目培养博士研究生1名，硕士研究生7名，本科毕业生20名。