<i>SmZIP11</i>基因过表达增强杨树对重金属的吸收和转运

杨钰颖; 陈舒鑫; 于淼; 贾瑜涵; 陆铸畴; 樊俐娇; 卓仁英; 韩小娇

doi:10.11674/zwyf.2023511

摘要:

目的利用木本植物修复重金属污染土壤是经济、安全、有效的方法。研究锌铁转运蛋白家族中SmZIP11基因表达对吸收和转运重金属的影响，为重金属污染土壤植物修复提供重要的基因资源。

方法采用馒头柳(Salix matsudana var. matsudana f. umbraculifera Rehd.)无性系一年生幼苗进行水培试验。幼苗在正常营养液中生长60天后进行重金属胁迫处理，重金属处理包括：200 µmol/L ZnSO₄、100 µmol/L CuSO₄和100 µmol/L CdSO₄。在重金属处理0、1、4、7、14和21天时，取根、茎和叶组织样品提取RNA，利用实时荧光定量PCR (quantitative real-time PCR, qRT-PCR)测定SmZIP11的表达水平。利用叶盘法将提取的SmZIP11基因转入银灰杨(Populus × canescens)中，经卡那霉素抗性筛选共得到8个过表达SmZIP11基因的转化杨树株系(OEs)。选择SmZIP11基因表达水平较高的3个转基因株系和野生型株系(WT)于重金属胁迫营养液中培养14天后，测定根、茎、叶生物量和重金属含量，计算耐受性指数和转移系数。

结果 SmZIP11基因编码区序列长度为1053 bp，编码351个氨基酸，分子量为37.71 kDa，含有9个保守的跨膜结构域，该基因编码的蛋白定位于细胞膜。系统进化树分析结果显示，馒头柳SmZIP11与杨柳科的亲缘关系最近，与大戟科、锦葵科、伞形科植物的ZIP11亲缘关系相对较远。qRT-PCR结果表明，在Zn、Cu和Cd胁迫下，馒头柳茎和叶片中的SmZIP11比对照显著上调；在Cd和Zn胁迫下，根、茎和叶片中该基因的表达量都显著提高。在转基因杨树中，SmZIP11基因显著增强了植株对Zn和Cu的耐受性，促进了Cd从根系向茎中转移，以及Zn向叶片中转移，同时将Cu富集在根部。与WT相比，OE7株系对Zn的耐受指数和地上部Zn的含量及转移系数都显著提高。

结论馒头柳SmZIP11基因增强了转基因杨树对Cu和Zn的耐受性，并提高了从根系向地上部转移Cd和Zn的能力，为植物修复土壤重金属污染提供了基因资源和理论指导。

Abstract:

Objective Woody plants are cost-effective and friendly sources for the remediation of heavy metal-contaminated soils. Investigating the function and the effect of gene SmZIP11, a member of zinc-regulated transporter and iron-regulated transporter-like protein family, on the uptake and translocation of heavy metals will provide valuable gene resources for plant remediation strategies.

Methods The 60-day-old rooted cuttings of willow (Salix matsudana var. matsudana f. umbraculifera Rehd.) were subjected to nutrient solutions containing 200 µmol/L ZnSO₄, 100 µmol/L CuSO₄, and 100 µmol/L CdSO₄, respectively. At the 0, 1, 4, 7, 14, and 21 days of stress, root, stem, and leaf samples were collected for the RNA extraction and determination of the expression levels of SmZIP11 by quantitative real-time PCR (qRT-PCR). Then, the SmZIP11 genes were transfected into Populus × canescens through the leaf disk method, and eight transgenic Populus × canescens plants (OEs) were constructed. Three OE plants with high SmZIP11 expression levels and the wild Populus × canescens plant (WT) were subjected to the heavy metal stress for 14 days, then the biomass and heavy metal contents were determined for the calculation of tolerance index and translocation factor.

Results The coding region of the SmZIP11 gene spans 1053 bp, encoding 351 amino acids with a molecular weight of 37.71 kDa. It harbors nine conserved transmembrane domains, and encoded protein was located in the cell membrane. Phylogenetic analysis revealed that SmZIP11 was closely related to Salicaceae, while showing more distant relationship to ZIP11 in Euphorbiaceae, Malvaceae, and Umbelliferaceae. SmZIP11 expression in leaves and stems were up-regulated under Zn, Cu, and Cd stress compared to the control. Meanwhile, the expression levels of SmZIP11 in roots, stems, and leaves showed a significant increase under Cd and Zn stress. In transgenic poplars, the SmZIP11 gene significantly enhanced the tolerance and transfer capabilities of Zn and Cu, facilitated Cd transfer from roots to stems and Zn to leaves, and accumulated Cu in roots. Compared to WT, OE7 plants exhibited higher tolerance to Zn, as well as Zn concentration and translocation factor in the aboveground parts.

Conclusion SmZIP11 enhanced tolerance to Cu and Zn, and improved the ability to transfer Cd and Zn from the root system to the aboveground parts. These findings will provide genetic resources and theoretical guidance for phytoremediation of soil heavy metal pollution.

SmZIP11基因过表达增强杨树对重金属的吸收和转运

Overexpression of SmZIP11 gene can enhance the absorption and transfer of heavy metals in poplar