控制大豆种子镉积累基因（GmHMA3）的功能分析

Soybean [Glycine Max (L.) Merr] has long been a staple food for Asian, containing rich plant protein and oil. The Food and Agriculture Organization and the World Health Organization proposed an upper limit of 0.2 mg/kg Cd concentration in soybean grain. There are cultivar differences in seed Cd accumulation in soybean and many cultivars accumulate high Cd concentrations in seed when grown on Cd-contaminated soils. Although there is no information available to show Cd in soybean has caused any adverse effects in humans, limiting Cd concentrations in edible produce and reducing the amount of Cd in the food chain are important human health issues. GmHMA3w cloned from a low seed Cd accumulator, acts as a Cd and Zn transporter in yeast; GmHMA3a cloned from a high seed Cd accumulator, has lost its function due to a SNP. In this program, we will use transgenic functional analysis, western-blot analysis, immunohistological staining, and subcellular localization in onion epidermal cells to insulate: (1) the different function between GmHMA3a and GmHMA3w in soybean; (2) the functional place of GmHMA3; (3) the function of GmHMA3 and the transport effect under the over-expression of GmHMA3w; (4) the model of Cd transport and storage in soybean; which will serve the low Cd accumulated breeding and the evaluate of soybean resources.

大豆是重要的粮食作物。我国2/3的大豆依赖进口，严重威胁我国的粮食安全和民生保障。不同大豆品种之间种子的镉积累能力不同，大部分现有栽培品种能够积累超过安全大豆标准（0.2 mg/kg干种子）的镉含量在种子中，潜在威胁人类健康。来源于大豆种子低镉积累品种的GmHMA3w基因在酵母细胞中具有转运镉和锌的能力，而高镉积累品种的GmHMA3a由于单碱基突变丧失了该功能。本项目利用转基因功能验证、蛋白质表达、免疫组化染色以及洋葱表皮细胞亚定位等对这两个基因进行深入研究，从而探讨：（1）GmHMA3a和GmHMA3w基因之间在大豆自身体内的功能差异；（2）GmHMA3基因的作用场所；（3）GmHMA3基因功能以及在超表达情况下GmHMA3w基因的功能效应；（4）镉在大豆体内的转运和储藏途径。为培育和选育低镉大豆品种以及大豆育种资源的评价提供理论资料。

控制大豆种子低镉积累候选基因GmHMA3w在酵母中转运镉和锌，而GmHMA3a由于单碱基突变丧失了该功能。但在大豆体内其生物学功能未知。本项目研究中，基因敲除GmHMA3w显著降低了根和叶片中的镉浓度，增加了大豆镉的耐受性。超表达GmHMA3w显著增加了根中镉浓度，但降低了茎秆中的镉浓度，对叶片中的镉浓度无影响，同时也增加了大豆镉的敏感性。酵母遗传转化表明GmHMA3w不转运钴、铁和铅；超表达表明该基因也不转运锌、镁和钙。超表达GmHMA3w显著降低细胞壁组份的镉浓度，但同时显著增加细胞器组份中的镉浓度，对细胞可溶性组份的镉浓度无影响。免疫组织化学染色和原生质体亚细胞定位显示GmHMA3w和3a位于细胞膜和细胞器（叶绿体除外）。这些结果证明GmHMA3w在大豆体内只转运镉，将大豆细胞壁积累的镉转运到细胞器中，并导致镉中毒以及降低生物学产量，从而阻碍大豆根部的镉向地上部分转运。