拟南芥耐受磷酸三苯酯毒性突变体的筛选及相关基因的功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1096

摘要/Abstract

摘要：

目的磷酸三苯酯（triphenyl phosphate, TPHP）是一种常见的环境有机污染物，威胁人类健康和生态安全。明确拟南芥耐受TPHP毒性的分子机制，可为后续利用植物修复土壤TPHP污染提供参考。方法以拟南芥野生型Col-0为实验材料，进行TPHP暴露处理，分析其根长、鲜重和叶绿素含量等表型指标的变化，利用显著变化的表型从拟南芥突变体库中筛选耐受TPHP毒性的突变体。之后，利用图位克隆和基因组重测序分析挖掘相关候选突变基因，并利用外源激素处理进行验证。最后通过分子对接、过表达株系和突变体分析，探究拟南芥耐受TPHP毒性的潜在分子机制。结果经2 mg/L TPHP处理后，拟南芥野生型Col-0幼苗的根长、鲜重和叶绿素含量均显著降低。根据表型变化筛选获得耐受TPHP毒性的突变体ems-29d（ems-29 dwarf）。该突变体植株矮小，叶片深绿皱缩。重测序分析发现，该突变体中参与油菜素内酯（brassinosteroids, BR）合成的关键基因DWF4，存在C1179T的单碱基变异，导致DWF4蛋白的第306位亮氨酸转变为苯丙氨酸（L306F）。外源施加表油菜素内酯（2,4-epibrassionolide, eBL）能部分恢复ems-29d的生长抑制表型。分子对接模拟结果显示，TPHP与DWF4^L306F的结合能小于与DWF4的结合能，但DWF4^L306F过表达转基因株系并未提高拟南芥对TPHP的耐受性，表明突变体的抗性并非靶标抗性。最后，经TPHP处理后，DWF4的T-DNA插入突变体dwf4叶绿素含量的受抑制程度显著低于野生型，可见BR合成受阻能显著提高拟南芥对TPHP的耐受性。结论 ems-29d中的DWF4基因突变，导致BR激素合成受阻，提高了植株对TPHP毒性的耐受。

关键词: 拟南芥, 磷酸三苯酯, 图位克隆, DWF4, 油菜素内酯, 分子对接, 过表达株系, 植物修复

Abstract:

Objective Triphenyl phosphate (TPHP) is a common environmental organic pollutant that poses threats to both human health and ecological security. Elucidation of the molecular mechanism underlying Arabidopsis thaliana tolerance to TPHP toxicity provides a reference for future phytoremediation strategies for TPHP-contaminated soil. Method Wild-type A. thaliana (Col-0) was used as the experimental material for TPHP exposure treatment and phenotypic analysis of root length, fresh weight, and chlorophyll content. A mutant with tolerance to TPHP toxicity was identified from an A. thaliana mutant library based on significantly altered phenotype. Map-based cloning and whole-genome resequencing analyses were then employed to identify the candidate mutant gene, with validation via exogenous hormone application. Finally, molecular docking, overexpression lines, and mutant analyses were conducted to preliminarily investigate the potential molecular mechanism of TPHP tolerance in A. thaliana. Result The root length, fresh weight, and chlorophyll content in wild-type Col-0 seedlings reduced significantly after treatment with 2 mg/L TPHP. A TPHP-tolerant mutant, designated ems-29d (ems-29 dwarf), was isolated based on phenotypic screening. This mutant was dwarf with dark green and wrinkled leaves. Resequencing analysis revealed the key gene DWF4 involved in brassinosteroids synthesis in mutants, containing C1179T single-nucleotide variation, resulting in a leucine-to-phenylalanine substitution at position 306 (L306F) in the DWF4 protein. Exogenous application of 2,4-epibrassinolide (eBL) partially restored the growth inhibition phenotype of ems-29d. Molecular docking simulations revealed a lower binding energy between TPHP and the mutant DWF4^L306F compared to that with the wild-type DWF4. However, the overexpression of DWF4^L306F in transgenic lines did not enhance TPHP tolerance, indicating that the resistance was not target-site mediated. Finally, following TPHP treatment, the suppression of chlorophyll content was significantly milder in the DWF4 T-DNA insertion mutant dwf4 than in the wild-type, demonstrating that impairment of BR biosynthesis markedly enhances A. thaliana tolerance to TPHP. Conclusion The mutation in DWF4 in ems-29d disrupts brassinosteroids synthesis, thereby increasing plant tolerance to TPHP toxicity.

Key words: Arabidopsis thaliana, triphenyl phosphate, map-based cloning, DWF4, brassinosteroid, molecular docking, overexpression line, phytoremediation

范帆, 李梦娇, 杨贤鹏, 崔莉莉. 拟南芥耐受磷酸三苯酯毒性突变体的筛选及相关基因的功能分析[J]. 生物技术通报, 2026, 42(7): 226-235.

FAN Fan, LI Meng-jiao, YANG Xian-peng, CUI Li-li. Screening and Functional Analysis of a Triphenyl Phosphate-tolerant Mutant in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2026, 42(7): 226-235.

图/表 9

表1 控制ems-29d矮小表型基因的精细定位

Table 1 Fine mapping of the gene controlling the dwarf phenotype of ems-29d

Sample	3‒11.22 M	3‒14.95 M	3‒17.36 M	3‒17.857 M	3‒18.89 M	3‒19.131 M	3‒19.679 M	3‒20.575 M	3‒20.8 M
1	A	A	A	A	A	A	A	A	H
2	A	A	A	A	A	A	H	H	H
3	A	A	A	A	A	A	H	H	H
7	A	A	A	A	A	H	H	H	H
8	H	H	H	H	A	H	H	H	H
12	H	H	H	H	A	A	A	A	A
16	H	H	H	H	A	H	H	H	H
18	A	A	A	A	A	A	H	H	H
19	B	B	H	H	A	A	A	A	A
21	A	A	A	A	A	A	A	H	H
24	H	H	H	H	A	A	A	A	A
27	H	H	H	H	A	A	A	A	A
32	H	H	H	A	A	A	A	A	A
33	A	A	A	A	A	A	A	A	H
36	H	H	A	A	A	A	H	H	H

图1 在含有TPHP的培养基上生长7 d的拟南芥野生型Col-0和Ler-0A：表型；B：根长；C：单株鲜重；D：叶绿素含量。不同小写字母表示样本间差异显著（P<0.05），下同

Fig. 1 Wild-type Col-0 and Ler-0 of A. thaliana grown on the medium containing TPHP for 7 dA: Phenotype. B: Root length. C: Fresh weight per plant. D: Chlorophyll content. The different lowercase letters indicate significant differences among samples (P<0.05). The same below

图2 TPHP处理7 d的拟南芥野生型Col-0和ems-29突变体A：表型；B：根长；C：单株鲜重；D：叶绿素含量。*指示ems-29d突变体

Fig. 2 Wild-type Col-0 and ems-29 mutant of A. thaliana after 7 d of TPHP treatmentA: Phenotype. B: Root length. C: Fresh weight per plant. D: Chlorophyll content. * indicates the ems-29d mutant

图3 ems-29d突变体DWF4基因的初定位*为Col-0条带，向右依次为ems-29突变体条带，Ler-0条带

Fig. 3 Preliminary mapping of the DWF4 gene in the ems-29d mutant* indicates the Col-0 band, moving to the right are the ems-29 mutant band and the Ler-0 band, respectively

图4 ems-29d中DWF4基因突变位点分析A：DWF4基因结构模式图；B：ems-29d中DWF4基因的突变位点

Fig. 4 Analysis of mutation sites of DWF4 gene in ems-29dA: Schematic representation of DWF4 gene structure. B: Mutation site on DWF4 gene in ems-29d

图5 0.1 μmol/L eBL处理4 d后的拟南芥野生型Col-0和ems-29d突变体A：表型；B：根长；C：单株鲜重

Fig. 5 Wild-type Col-0 and ems-29d mutant of A. thaliana after 4 d of treatment with 0.1 μmol/L eBLA: Phenotype. B: Root length. C: Fresh weight per plant

图6 TPHP与DWF4（A）及DWF4L306F（B）相互作用分析

Fig. 6 Interaction analysis of TPHP with DWF4 (A) and DWF4L306F (B)

图7 TPHP处理7 d后的拟南芥野生型Col-0和DWF4L306F OE转基因株系A：DWF4L306F OE转基因株系的分子鉴定；B‒E：2 mg/L TPHP处理后拟南芥野生型Col-0和DWF4L306F OE转基因株系的表型（B）、根长（C）、单株鲜重（D）和叶绿素含量（E）

Fig. 7 Wild-type Col-0 and DWF4L306F transgenic lines of A. thaliana after 7 d of TPHP treatmentA: Molecular identification of DWF4L306F transgenic lines. B-E: Phenotypes (B), root length (C), fresh weight per plant (D) and chlorophyll content (E) of wild-type Col-0 and the DWF4L306F OE transgenic lines of A. thaliana after treatment with 2 mg/L TPHP

图8 在含有不同浓度TPHP的培养基上生长7 d的拟南芥野生型Col-0和dwf4突变体A：表型；B：叶绿素含量；C：叶绿素含量抑制率。*、**和***分别表示在P<0.05、P<0.01和P<0.001水平差异显著

Fig. 8 Wild-type Col-0 and dwf4 mutants of A. thaliana grown for 7 d on media containing different concentrations TPHPA: Phenotype. B: Chlorophyll content. C: Chlorophyll content inhibition rate. *, **, and *** indicate significant differences at P<0.05, P<0.01, and P<0.001 levels, respectively

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