OsLCT1启动子驱动OsNRAMP5表达对镉向水稻种子迁移的影响

doi:10.3864/j.issn.0578-1752.2025.07.001

中国农业科学 ›› 2025, Vol. 58 ›› Issue (7): 1259-1268.doi: 10.3864/j.issn.0578-1752.2025.07.001

• 作物遗传育种·种质资源·分子遗传学 • 下一篇

OsLCT1启动子驱动OsNRAMP5表达对镉向水稻种子迁移的影响

熊嘉妮¹(), 李宗玥¹(), 胡衡亮¹, 顾天宇¹^,², 高艳¹^,², 彭佳师¹^,²()

¹ 湖南科技大学生命科学与健康学院，湖南湘潭 411201
² 湖南科技大学/经济作物遗传改良与综合利用湖南省重点实验室/重金属污染土壤生态修复与安全利用湖南省高校重点实验室，湖南湘潭 411201

收稿日期:2024-08-06 接受日期:2024-10-04 出版日期:2025-04-08 发布日期:2025-04-08
通信作者:
彭佳师，E-mail：jspeng@hnust.edu.cn
联系方式: 熊嘉妮，E-mail：2948345918@qq.com。李宗玥，E-mail：331439387@qq.com。熊嘉妮和李宗玥为同等贡献作者。
基金资助:
国家自然科学基金(U20A2024); 国家自然科学基金(32400252); 湖南省自然科学基金(2022JJ20022); 湖南省自然科学基金(2024JJ6213); 湖南省“三尖”创新人才工程(2023RC3177)

Influence of Expressing OsNRAMP5 Under the Driving of the OsLCT1 Promoter on Cadmium Migration to Rice Seeds

XIONG JiaNi¹(), LI ZongYue¹(), HU HengLiang¹, GU TianYu¹^,², GAO Yan¹^,², PENG JiaShi¹^,²()

¹ School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan 411201, Hunan
² Hunan University of Science and Technology/Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization/Hunan Province University Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-Polluted Soils, Xiangtan 411201, Hunan

Received:2024-08-06 Accepted:2024-10-04 Published:2025-04-08 Online:2025-04-08

摘要/Abstract

摘要：

【目的】镉（Cd）是我国耕地的主要污染物，而稻米是人体Cd摄入的主要来源。OsNRAMP5是介导Cd进入水稻体内的主要转运蛋白，在水稻特定组织中特异表达OsNRAMP5，探索并构建种子低积累Cd水稻的途径，为应对耕地Cd污染水稻改良的分子设计提供参考。【方法】选取OsLCT1起始密码子上游2 500 bp序列作为启动子，驱动OsNRAMP5在水稻日本晴中表达，并通过在OsNRAMP5的C端融合红色荧光蛋白mRFP以观察OsNRAMP5在转基因水稻中的组织表达情况。获得独立的纯合转基因株系后，首先用qRT-PCR检测OsNRAMP5在水稻中的表达，利用激光共聚焦显微镜观察OsNRAMP5在水稻根和节点处的组织定位；其次，观察和统计野生型和转基因株系在不同浓度Cd处理条件下对Cd的积累和耐受特性的变化；最后，在含Cd污染的农田土壤中种植，统计Cd和其他矿质元素在种子和叶片中的积累及产量性状。【结果】在OsLCT1启动子驱动下，OsNRAMP5主要在水稻根部的表皮、外皮层、中柱，以及节中的扩大维管束韧皮部和扩散维管束中表达，这与水稻OsNRAMP5的原有表达模式显著不同。与野生型相比，在幼苗期，转基因株系表现出根部Cd积累增加，地上部Cd积累减少的表型，而且对Cd胁迫的耐受性增强；在含Cd污染的农田土壤中种植后，转基因株系的株高产量等没有变化，但种子和叶片中Cd的积累显著减少。其中，种子中Cd的积累降低约80%，减少的比例远大于叶片。地上部和种子中Mn的含量也略有减少，但是Fe、Zn、Cu等矿质元素积累变化不显著。【结论】OsLCT1启动子驱动OsNRAMP5在水稻中表达，减少了Cd从根部向地上部转运，同时，在节点部位使Cd更多地分配至叶片，从而进一步降低了种子中Cd的积累。因此，利用OsLCT1启动子驱动OsNRAMP5表达是降低水稻种子Cd积累的有效方式。

关键词: 镉, OsLCT1, OsNRAMP5, 特异表达, 修复型水稻, 低镉水稻

Abstract:

【Objective】Cadmium (Cd) is the predominant pollutant in China’s arable land, with rice cultivated on these contaminated soils being a significant dietary source of Cd for the population. This study aims to tissue-specifically express OsNRAMP5, a transporter responsible for the majority of Cd uptake in rice, to investigate strategies for developing low-Cd rice varieties and provide a reference for molecular design breeding to cope with Cd pollution. 【Method】To drive the expression of OsNRAMP5 in rice, we utilized a 2 500 bp sequence upstream of the OsLCT1 start codon as the promoter. The red fluorescent protein mRFP was fused to the C-terminus of OsNRAMP5 to visualize its tissue localization. After obtaining independent homozygous transgenic lines, the transcripts of the OsNRAMP5 were first detected using qRT-PCR, and its tissue localization in roots and nodes was observed via laser confocal microscopy. Subsequently, the accumulation and tolerance of Cd were evaluated in transgenic and wild-type rice under varying concentrations of Cd treatment. Furthermore, plants were grown in Cd-contaminated paddy soil, and the accumulation of Cd and other mineral elements in seeds and leaves, as well as related yield traits, were measured. 【Result】Under the drive of the OsLCT1 promoter, OsNRAMP5 was expressed mainly in the epidermis, exodermis and stele of roots, as well as in the phloem area of enlarged vascular bundles and diffuse vascular bundles in nodes, differing significantly from the native expression pattern of OsNRAMP5 in rice. Compared to wild-type rice, the transgenic lines exhibited increased Cd accumulation in roots, decreased Cd accumulation in shoots, and enhanced tolerance to Cd stress during the seedling stage. When cultivated in Cd-contaminated paddy soils, plant height and grain yield were unaffected by the ectopic expression of OsNRAMP5, while Cd accumulation in seeds and leaves significantly decreased in the transgenic lines. The Cd content in seeds decreased by over 80%, with a greater reduction ratio compared to that in leaves. Although the Mn content in seeds and leaves slightly decreased, the expression of OsNRAMP5 had little impact on the accumulation of other mineral elements such as Fe, Zn, and Cu. 【Conclusion】The expression of OsNRAMP5 driven by the OsLCT1 promoter greatly decreases the Cd migration toward rice seeds by reducing Cd transport to the aboveground parts from roots and increasing the Cd transporting to leaves at nodes. Therefore, the expression of OsNRAMP5 under the control of the OsLCT1 promoter is an effective strategy to reduce Cd accumulation in rice seeds.

Key words: cadmium, OsLCT1, OsNRAMP5, specific expression, remediation-rice, low-cadmium rice

熊嘉妮, 李宗玥, 胡衡亮, 顾天宇, 高艳, 彭佳师. OsLCT1启动子驱动OsNRAMP5表达对镉向水稻种子迁移的影响[J]. 中国农业科学, 2025, 58(7): 1259-1268.

XIONG JiaNi, LI ZongYue, HU HengLiang, GU TianYu, GAO Yan, PENG JiaShi. Influence of Expressing OsNRAMP5 Under the Driving of the OsLCT1 Promoter on Cadmium Migration to Rice Seeds[J]. Scientia Agricultura Sinica, 2025, 58(7): 1259-1268.

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OsLCT1启动子驱动OsNRAMP5表达对镉向水稻种子迁移的影响

Influence of Expressing OsNRAMP5 Under the Driving of the OsLCT1 Promoter on Cadmium Migration to Rice Seeds

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