萝卜（Raphanus sativus L.）是十字花科萝卜属重要的根茎类蔬菜，营养丰富，食 疗价值高。萝卜肉质根易富集重金属离子，并可通过食物链在人体内富集，威胁人类健康。镉（Cd）是生物毒性极强的重金属之一。土壤Cd污染是萝卜高品质栽培与安 全生产的重要限制因素，如何有效控制萝卜等根菜类蔬菜Cd吸收累积是蔬菜遗传育 种领域重要研究课题。天然抗性相关巨噬细胞蛋白（Natural Resistance-Associated Macrophage Protein, NRAMP）是一类重要的金属离子转运蛋白，其包含10~12个跨膜 结构域，可介导植物体内Cd、铅（Pb）、锌（Zn）、锰（Mn）等金属离子吸收转运过 程。然而，萝卜中RsNRAMPs基因尚未分离鉴定，其调控萝卜肉质根Cd吸收累积的 分子通路仍不明确，极大地阻碍了萝卜低Cd吸收累积性状遗传改良与种质创新利用 进程。本研究在萝卜全基因组水平分离RsNRAMPs基因家族成员，分析其在不同发育 时期及重金属胁迫下的时空表达模式，通过酵母异源转化分析RsNRAMP5基因金属离 子转运活性，分析RsNRAMP5基因调控萝卜肉质根Cd吸收累积及胁迫响应的作用机 制；运用酵母单杂交（Y1H）和双荧光素酶（DLA）技术，鉴定出RsNRAMP5基因上 游调控因子RsCDF3，解析RsCDF3转录因子介导RsNRAMP5基因调控萝卜Cd吸收 累积的分子机制。主要研究结果如下： 1、基于萝卜‘NAU-LB’基因组数据库，共鉴定出9个RsNRAMPs基因成员，分为 三个亚组，不均匀分布于萝卜 7 条染色体上。启动子顺式作用元件分析发现， RsNRAMPs 基因可参与萝卜生长发育、离子运输、激素信号和胁迫响应等生物过程。 RNA-seq分析表明，与幼苗期相比，6个RsNRAMPs基因（RsNRAMP1.1、RsNRAMP2.1、 RsNRAMP2.2、RsNRAMP3、RsNRAMP4 和 RsNRAMP5）在破肚期和肉质根膨大期均 表达上调，而RsNRAMP1.2和RsNRAMP6表达下调。RT-qPCR分析表明，RsNRAMP3、 RsNRAMP4 和 RsNRAMP5 在维管形成层中高水平表达。50 μM CdCl2 处理下， RsNRAMP1.1仅在24 h表达上调，而RsNRAMP3在6 h和24 h上调表达，RsNRAMP1.3、 RsNRAMP2.1、RsNRAMP4 和 RsNRAMP5 在各个时间点均显著上调表达。其中， RsNRAMP5 基因在维管形成层和Cd胁迫24 h表达量最高，表明其可能在萝卜肉质根 发育与Cd吸收累积中发挥重要作用。

2、分离出萝卜RsNRAMP5基因，CDS为1662bp，编码553个氨基酸，包含10 个跨膜结构域。RsNRAMP5定位于细胞膜，其启动子活性受Cd胁迫显著诱导。25 μM 和50 μM CdCl2处理下，过表达RsNRAMP5基因酵母菌株长势明显弱于转化空载的酵 母，并可促进酵母细胞对Cd、Pb、Fe、Mn的累积，而转化RsNRAMP3和RsNRAMP4 对酵母细胞生长影响不显著。50 μM CdCl2处理下，过表达RsNRAMP5基因的T3代拟 南芥株系OE-2与OE-4根中Cd累积量与活性氧（ROS）含量显著增加。遗传转化分 析发现，RsNRAMP5-OE萝卜子叶中Cd含量比对照组增加，RsNRAMP5-RNAi子叶中 Cd 含量下降；RsNRAMP5-OE子叶中H2O2、O2·−和MDA含量显著高于对照组，脯氨 酸（Pro）含量下降，而 RsNRAMP5-RNAi 萝卜子叶中相关指标表现出相反趋势。 RT-qPCR 分析发现，50 μM CdCl2处理下，RsNRAMP5-OE萝卜子叶中ROS清除相关 基因（RsCAT2、RsCAT3、RsAPX 和RsSOD）及 Pro产生相关基因（RsP5CR、RsP5CDH、 RsP5CS1和RsP5CS2）均显著下调表达，而在RsNRAMP5-RNAi萝卜子叶中表达上调， 进而导致RsNRAMP5基因介导的ROS积累和Pro含量升高。 3、通过酵母单杂筛选到RsCDF3转录因子可直接结合RsNRAMP5基因启动子， RsCDF3 特异结合RsNRAMP5 基因启动子-362~-355bp 处 DOF 元件（AAAAAGTA）， 该元件突变为AAAAAAAA时酵母显色板上蓝色消失。DLA实验表明，RsCDF3抑制 RsNRAMP5 基因的转录水平。RsCDF3 定位于细胞核，其启动子活性受Cd胁迫显著 诱导。酵母及烟草转化发现，RsCDF3是转录抑制因子，其N端与C端均未发现转录 激活域。通过农杆菌转化获得T3代RsCDF3转基因拟南芥植株，在含有50 μM CdCl2 的MS培养基上，RsCDF3-OE-1、RsCDF3-OE-4 植株根长比 WT 显著增长，其 ROS 与MDA累积量显著低于WT。进一步利用双硫腙染色和Cd含量测定分析，均证实 RsCDF3转录因子通过抑制RsNRAMP5基因转录，降低拟南芥植株对Cd的吸收累积， 解析了RsCDF3-RsNRAMP5转录模块调控萝卜镉吸收累积的分子机制。

Radish (Raphanus sativus L.) is an important root vegetable belonging to the Brassicaceae family. The radish taproot is rich in nutrition and has high medicinal therapeutic value. The heavy metal ions are easily taken up by radish taproots, which can also enter human body through the food chain, thereby threatening human health. Cadmium (Cd) is one of the most toxic heavy metals. Soil pollution of Cd has becoming an vital limiting factor for high-quality cultivation and safe production in radish. How to control the Cd uptake and accumulation in radish and other root vegetables have enmerged as an important research topic in the field of vegetable genetic breeding. Natural Resistance-Associated Macrophage Protein (NRAMP) is an important class of metal ion transporters, which contains 10~12 transmembrane domains. Many NRAMPs can mediate the absorption and transport of Cd, lead (Pb), zinc (Zn), manganese (Mn) and other metal ions in plants. However, the RsNRAMP genes have not been isolated and identified, and the molecular regulatory pathways of root Cd uptake and accumulation are still unclear in radish, which greatly impedes the process of genetic improvement as well as low-Cd-content germplasm innovation and utilization in radish. In this study, we firstly isolated the RsNRAMP gene members at the whole radish genome level, and investigated their spatial and temporal expression patterns in different developmental stages and under heavy metal stress. Moreover, we explored the metal transport activity of RsNRAMP5 through yeast heterologous transformation, and investigated the critical role of RsNRAMP5 in the regulatory pathways of of Cd uptake and accumulation in radish. Furthermore, we found that the RsCDF3 can directly bind to the RsNRAMP5 promoter using the yeast one-hybrid (Y1H) and dual-luciferase (DLA) approach, and clarified the molecular mechanism underlying RsCDF3-RsNRAMP5 mediated Cd uptake and accumulation in radish. The major results of the study are as follows:

1. Based on the radish ‘NAU-LB’ genome database, a total of nine RsNRAMP gene members were identified, which were categorized into three subgroups and unevenly distributed on the seven radish chromosomes. The promoter cis-acting element analysis revealed that these RsNRAMPs could involved in several biological processes such as growth and development, ion transport, hormone signaling and stress response in radish. RNA-seq analysis showed that six RsNRAMPs (RsNRAMP1.1 、 RsNRAMP2.1 、 RsNRAMP2.2、RsNRAMP3、RsNRAMP4 and RsNRAMP5) were up-regulated in cortex splitting stage and expanding stage compared with pre-cortex splitting stage, whereas both the RsNRAMP1.2 and RsNRAMP6 were down-regulated under the same comparison. RT-qPCR analysis showed that the RsNRAMP3, RsNRAMP4 and RsNRAMP5 exhibited high expression in the vascular cambium of radish root. Under 50 μM CdCl2 treatment, RT-qPCR analysis showed that the expression of RsNRAMP1.1 was up-regulated only at 24 h, whereas RsNRAMP3 exhibited up-regulated at 6 h and 24 h. The RsNRAMP1.3, RsNRAMP2.1, RsNRAMP4 and RsNRAMP5 exhibited significant induced expression at three time points under 50 μM CdCl2 treatment. Among them, the RsNRAMP5 had the highest expression level in the vascular cambium and under CdCl2 treatment, suggesting that the RsNRAMP5 gene might play an important role in root Cd uptake and accumulation in radish. 2. The CDS of RsNRAMP5 gene was 1662 bp, encoding 553 amino acids and containing 10 transmembrane domains. The RsNRAMP5 was localized in the cell membrane and its promoter activity was significantly induced by Cd stress. Yeast transformation analysis showed that the growth of RsNRAMP5-overexpressing yeast strains were significantly weaker than yeast transformed with the empty vector under 25 μM and 50 μM CdCl2 treatments. It also promoted the accumulation of Cd, Pb, Fe and Mn in yeast cells, whereas the transformation of RsNRAMP3 and RsNRAMP4 had no significant effect on the yeast cell growth. Heterologous transformation analysis revealed that overexpression of RsNRAMP5 significantly increased Cd accumulation and reactive oxygen species (ROS) content in the roots of positive T3-generation Arabidopsis thaliana lines OE-2 and OE-4 under 50 μM CdCl2 treatment. Using the transient transformation technique, we found that the Cd content was increased in RsNRAMP5-OE radish cotyledons compared with the control, which was decreased in the RsNRAMP5-RNAi cotyledons. The content of H2O2,O2·− and MDA were significantly higher in the RsNRAMP5-OE cotyledons compared with the control, and proline content was decreased, whereas the related indexes showed an opposite trend in the RsNRAMP5-RNAi radish cotyledons. RT-qPCR analysis found that 50μM CdCl2 treatment, four ROS-scavenging related genes (RsCAT2, RsCAT3, RsAPX and RsSOD) and proline production related genes (RsP5CR, RsP5CDH, RsP5CS1 and RsP5CS2) were significantly down-regulated in the RsNRAMP5-OE radish cotyledons. However, the expression of these genes was up-regulated in RsNRAMP5-RNAi radish cotyledon, thereby leading to the RsNRAMP5-mediated inducution of ROS accumulation and proline content process. 3. The RsCDF3 transcription factor can directly bind to the RsNRAMP5 gene promoter by yeast one-hybrid approach. RsCDF3 specifically binds to the DOF element (AAAAAGTA) at -362~-355bp of the RsNRAMP5 gene promoter, and the blue color disappeared from the yeast plate when the binding element wass mutated to AAAAAAA. The dual luciferase assay showed that RsCDF3 repressed the transcription of the RsNRAMP5. Further analysis revealed that RsCDF3 was localized in the nucleus and its promoter activity was significantly induced by Cd stress. By heterologous transformation of yeast and tobacco plants, RsCDF3 was found to be a transcriptional repressor, and no transcriptional activation domains were found at its N-terminal and C-terminal ends. Two T3-generation RsCDF3 transgenic Arabidopsis plants (RsCDF3-OE-1 and RsCDF3-OE-4) were obtained by Agrobacterium transformation. Notably, the root lengths of both two RsCDF3-OE Arabidopsis lines , were significantly increased compared with the WT plants on MS medium containing 50μM CdCl2, and the ROS and MDA content was significantly lower than those of WT. Further dithizone assay and Cd content determination revealed that the RsCDF3 reduces the Cd uptake and accumulation in Arabidopsis plants by repressing the RsNRAMP5 transcription. These results would facilitate clarifying the molecular mechanism underlying RsCDF3-RsNRAMP5 mediated Cd uptake and accumulation in radish.

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