NtMTP蛋白在烟草Cd转运及分配过程中的功能分析

Heavy metal ions such as Cu2+, Zn2+, Mn2+, Fe2+, Ni2+ and Co2+ are essential micronutrients for plant metabolism but when present in excess, these, and non-essential metals such as Cd2+, Hg2+ and Pb2+, can become extremely toxic. Cadmium (Cd) can trigger many deleterious effects on plants such as the reduction in the leaf surface, dry weight, water content, chlorophyll content, and carotenoid content. One way Cd enters the human body is by ingestion of Cd-contaminated food. However, inhalation of smoke from cigarettes is a far more effective uptake route than absorption through the gastrointestinal tract. Exposure to Cd induces damage in bones, lungs and liver. Cd has also been reported to deregulate hormone balance leading to spontaneous abortion. It is thus of interest that tobacco plants accumulate less cadmium in the leaves so that less cadmium would be found in the smoke of cigarettes. .In recent years, with the development of modern biotechnology, scientists began trying to reduce the Cd concentration of tobacco leaves by gene manipulation. Because Cd is thought to be largely accumulated in the vacuole in tobacco under low and high Cd doses, two obvious strategies can be applied to root retention to reduce leaf accumulation. One is trapping Cd in the cytosol of root cells by chelation. Another one is enhancing the vacuolar storage/sequestration of Cd by over-expressing the membrane localized heavy metal transporters in tobacco roots. .There is a number of gene families involved in metal transport in plant, among which members of a CDF family of divalent cation transporters have been implicated in conferring heavy metal tolerance. Plant CDF transporters are designated metal-tolerance proteins (MTPs) and have been classified into three groups: zinc (Zn)-CDFs, iron (Fe)/Zn-CDFs, and manganese (Mn)-CDFs. It has been suggested that, besides the Mn-CDFs, members of the other two groups play a key role in Cd transport and accumulation. The tobacco genome encodes 6 putative cation-efflux proteins, which can be divided into three distinct groups (NtMTP1A, 1B, NtMTPC2 belong to Zn-CDFs; NtMTPC4 belongs to Fe/Zn-CDFs; NtMTP4 and 5 belong to Mn-CDFs). Nevertheless, only NtMTP1A and 1B have been functional studied. .In this study, the molecular and functional characterization of NtMTPC2 and NtMTPC4, including gene expression pattern and cellular protein localization will be studied in detail. Moreover, the tolerance to Cd and the accumulation of Cd in tobacco tissues will be investigated using plants expressing CaMV35S::NtMTP or root-specific promoter FS3::NtMTP, as well as artificial microRNA (amiRNA) constructs. All these results will shed some light on the biological functions of NtMTPC2 and C4 as divalent cation transporters in Cd transport, distribution and tolerance in tobacco. Furthermore, this study will be a good example for reducing the human Cd intake from tobacco via the enhancement of root vacuolar sequestration of this pollutan.

烟草对重金属Cd具有较强的吸收和转运能力，Cd污染不仅影响烟草的生长发育，同时也会降低烟叶的品质，对人体健康造成危害。相比传统的防控修复手段，利用基因操控的方法来控制烟草Cd污染已成为一种新的方向，但是目前对烟草重金属Cd响应的特异基因的研究还很少，基因资源较为缺乏。本项目拟以烟草重金属耐受蛋白编码基因NtMTPC2和NtMTPC4为研究对象，采用实时定量PCR、GUS活性测定、荧光显微观察和酵母突变体互补等方法，研究基因的表达模式、蛋白亚细胞定位以及对Cd的响应和转运特性，从而揭示NtMTP蛋白在烟草Cd转运及分配过程中的生物学功能。同时拟利用基因工程手段，获得两个NtMTP基因CaMV35S和根特异过量表达，以及人工合成miRNA的转基因烟草植株，检测转基因烟草叶片和根中Cd的含量以及对Cd污染的耐受性，为今后利用基因操控的方法控制烟草重金属Cd污染提供重要的基因资源和理论依据。

重金属可分为植物代谢必需的微量元素，如Zn、Fe、Mn，和非必需金属，如Cd，过量的必需元素和非必需金属都会对植物产生危害。烟草属于易累积重金属Cd的植物，Cd污染不仅影响烟草的生长发育，同时也会降低烟叶的品质，对人体健康造成危害。由于Cd不是植物必需的矿质元素，因此植物根部对Cd的吸收主要依靠Zn、Fe、Mn等与其化学结构相似的必需元素的转运蛋白或通道。金属耐受蛋白MTP是一类重要的膜定位二价阳离子转运蛋白，目前已在多个物种中被分离鉴定，但是在烟草中NtMTP基因的克隆和功能研究还鲜有报道，这些基因在烟草重金属响应、吸收和转运过程中的功能尚不明确。本项目首先采用生物信息学的方法从烟草基因组中鉴定到26个NtMTP基因，对这些基因的分类、序列特征、蛋白结构以及染色体位置等进行了综合分析，解析了烟草MTP基因家族的进化历程，预测了NtMTP基因启动子顺式作用元件的组成和分布，以及NtMTP基因编码区microRNA的靶位点，为NtMTP基因的表达调控研究提供了重要启示；通过利用烟草转录组数据和qRT-PCR分析，研究了NtMTP基因的组织表达模式，以及在不同重金属处理下的表达模式，为以后解析NtMTP基因的生物学功能提供了重要数据和理论基础。在此基础上，进一步以烟草NtMTP1、NtMTPC2、NtMTPC4和NtMTP5基因为对象，研究了它们在烟草重金属转运及分配过程中的生物学功能。发现：1）过量表达NtMTP1基因增强了烟草对Zn的耐受性；2）NtMTPC4启动子是一个花粉特异启动子，其编码蛋白可能定位于液泡膜上；3）NtMTPC2和NtMTPC4不能在酵母中转运Zn2+、Cd2+、Mn2+和Co2+；4）NtMTP5是一个定位于高尔基体上的Mn2+转运蛋白，该编码基因的表达具有组织特异性且在叶片中受到Mn2+的诱导。这些结果为全面解析NtMTP基因在烟草重金属响应、吸收和转运过程中的功能奠定了基础，为今后利用基因操控的方法控制烟草重金属污染提供了重要的基因资源和理论依据。