矿区野生苎麻镉富集性和耐性及分子标记与相关基因的克隆

The area of soil's heavy metals pollution were granted to 20 million hectares in China, accounting to 1/5 of the total arable land. Cd pollution is one of the most serious pollutions. Ramie (Boehmeria nivea(L.) Gaud.) has the advantages such as high biomass, strong heavy metal tolerance,and not bringing toxic metals into food chains, which can be applied in the restoration of heavy metal pollution with high economic benefit.The study showed that wild ramie is a common plant growing in mining area with strong tolerance and high bioaccumulation to multi heavy metals, which is probably Cd and Pb hyper-accumulator. In the basis of completing the identification and evaluation of Cd tolerance and accumulation characteristics of wild ramie germplasm resourses, our research group accomplished the jobs as follows, the transcriptome and expression profile sequencing analysis of ramie under Cd Stress ,abtaining 3000 EST possess the characteristic of SSR and 3000 genes relative to Cd stress , analysis of Cd tolerance molecular mechanism , the isolation and expression of important genes, screening of polymorphic SSR primers. The 96 wild ramies which had been identificated and evaluated on tolerance and accumulation characteristic of Cd will be chosed to do molecular marker，association analysis，gene clone and expression characteristic. These jobs will provid theory and technique foundation for tolerance and accumulation mechanism study of heavy metal strees on ramie. And it will contribute to marker-assisted selection breeding and Gene Engineering Breeding of ramie.

我国重金属污染土壤已达2000万hm2，约占总耕地面积的1/5，镉污染是较严重污染之一。苎麻生物量大、耐重金属能力强且产品不进入食物链，既可以修复重金属污染土壤，又可获得较好经济效益。研究表明：野生苎麻是我国南方金属矿区分布的优势植物，不仅能耐和富集多种重金属，而且可能是镉、铅的超富集植物。在完成典型矿区野生苎麻种质镉耐性和富集特征初步鉴定评价的基础上，申请者课题进行了苎麻转录组测序和镉胁迫苎麻表达谱测序，获得了3000多个具有SSR特征的EST序列和3000多个镉胁迫相关的基因，初步分析了苎麻耐镉的的分子机理，开展了重要相关基因的表达分析、克隆，开展了SSR引物多态性筛选，选择经鉴定评价的镉耐性和富集性的96份矿区野生苎麻进行耐性、富集性的分子标记、关联分析、重要基因克隆与表达分析，为苎麻耐和富集重金属机理研究和分子标记辅助育种与基因工程育种提供理论与技术基础。

苎麻生物量大、耐重金属能力强且产品不进入食物链，既可以修复重金属污染土壤，又可获得较好经济效益。本课题完成了典型矿区野生苎麻种质收集及镉耐性和富集特征的鉴定评价，获得了高耐或高积累Cd的苎麻野生种质34份，低耐或低积累Cd苎麻野生种质15份。基于苎麻转录组测序筛选苎麻EST-SSR引物447个，部分EST-SSR标记与苎麻镉耐性与富集性性状的关联分析，获得了与苎麻镉耐性与富集性标记3个；基于苎麻转录组测序、镉胁迫下苎麻表达谱测序获得镉胁迫响应基因3887个，其中上调基因占总数的74%。苎麻镉相关基因主要富集途径为谷胱甘肽代谢途径(Glutathione metabolism)以及半胱氨酸、甲硫氨酸代谢途径(Cysteine and methionine metabolism)。共计55个上调基因，其中，在谷胱甘肽代谢途径中上调表达的19个基因，在半胱氨酸和甲硫氨酸代谢途径中上调表达的36个基因，通过对其中16个重要基因在镉耐性与富集性不同的苎麻品种的根、茎、叶不同器官的表达分析，获得了苎麻镉胁迫响应基因8个；基于miRNA测序、转录组测序和镉胁迫表达谱测序相结合，筛选到响应镉胁迫相关基因2个：miR171,miR393。在此基础上，克隆了16个基因，分别为：BnAPX1、BnGR1、BnPCS1、BnbZIP1、BnbZIP2、BnbZIP3、BnMYB1、BnMYB2、BnMYB3、BnG6PDH、MAN、XTH1、XTH2、XTH3、XTH4和XTH5；构建了10个镉胁迫响应基因的植物表达载体、并转化根癌农杆菌，获得了各基因的转基因拟南芥植株，并证明BnPCS1、BnbZIP3、BnXTH1与镉耐性相关，为苎麻耐和富集重金属机理研究和分子育种提供了一定的理论与技术基础。