灰杨PcAHL17负调控拟南芥的镉耐受性

doi:10.13560/j.cnki.biotech.bull.1985.2025-0074

摘要/Abstract

摘要：

目的研究灰杨（Populus × canescens）PcAHL17（AT-hook motif nuclear localized proteins, AHLs）调控植物响应镉胁迫的分子机制，为抗逆良种的选育和生态修复的应用提供参考。方法以野生型（WT）、转空载体对照（VC）和过表达PcAHL17拟南芥（PcAHL17-OE1、PcAHL17-OE2和PcAHL17-OE3）为材料，研究PcAHL17调控植物响应镉胁迫的机制。结果镉处理后，灰杨根茎叶的PcAHL17表达量均有变化；过表达株系的萌发率和根长均明显低于WT和VC；过表达株系的抗氧化物酶的活性和转录水平均有所提升，但仍低于WT和VC；过表达株系细胞膜受损伤程度显著高于WT和VC，根部积累的Cd²⁺和H₂O₂含量和Cd²⁺内流显著高于WT和VC；过表达株系叶绿素含量、叶绿素荧光参数和光合速率等方面均低于WT和VC。结论过表达灰杨PcAHL17能够负调控拟南芥的镉耐受性。

关键词: 灰杨, 镉胁迫, AT-hook核定位蛋白, PcAHL17, 离子平衡, 活性氧

Abstract:

Objective We delved into the molecular mechanisms by which AHL17 (AT - hook motif nuclear - localized proteins, AHLs) in Populus×canescens regulates the plant’s response to cadmium stress. The findings of this research offered a reference for the breeding of stress-resistant varieties and the application of ecological restoration. Method Wild-type (WT), vector control (VC), and Arabidopsis thaliana overexpressing PcAHL17 (PcAHL17-OE1, PcAHL17-OE, and PcAHL17-OE3) were used as experimental materials, the mechanism of PcAHL17 regulating plant response to cadmium stress was studied. Result After cadmium treatment, the expressions of PcAHL17 in the roots, stems, and leaves of Populus×canescens changed. The germination rate and root length of the overexpressed lines showed a significant decrease compared to those of WT and VC. The activity and transcription level of antioxidant enzymes in the overexpressing strains both increased, but still were lower than those in WT and VC. The degree of membrane damage in the overexpressing strains was significantly higher than that in WT and VC, and the accumulation of Cd²⁺and H₂O₂ in roots, as well as the influx of Cd²⁺, were significantly higher than those in WT and VC. Overexpressed strains had lower chlorophyll content, chlorophyll fluorescence parameters, and photosynthetic rate than WT and VC. Conclusion The overexpression of PcAHL17 gene may negatively regulate tolerance of transgenic Arabidopsis thaliana to cadmium.

Key words: Populus × canescens, cadmium stress, AT-hook motif nuclear localized proteins, PcAHL17, ion homeostasis, reactive oxygen species

冯冰, 闫彩霞, 刘艺, 董凯悦, 赵楠, 赵瑞, 陈少良. 灰杨PcAHL17负调控拟南芥的镉耐受性[J]. 生物技术通报, 2025, 41(6): 269-283.

FENG Bing, YAN Cai-xia, LIU Yi, DONG Kai-yue, ZHAO Nan, ZHAO Rui, CHEN Shao-liang. Populus × canescens AHL17 Negatively Regulates Tolerance to Cadmium in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2025, 41(6): 269-283.

图/表 15

表1 本实验所用引物序列

Table 1 Primers used in this study

引物名称 Primer name	上游引物 Forward primer （5'-3'）	下游引物 Reverse primer （5'-3'）
PcUBQ	AGACCTACACCAAGCCCAAGAAGA	CCAGCACCGCACTCAGCATTAG
AtACTIN2	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCTGC
AtSOD1	AGGAAACATCACTGTTGGAGAT	GAGTTTGGTCCAGTAAGAGGAA
AtPER1	CGTGCCCTTCATATTGTTGG	GACGCCATCAACAACGAGTC
AtCAT3	CTTGTGGTTCCTGGAATCTACT	AGGATCAAACTTTGAGGGGTAG
PcAHL17	GGGGCCCGGGGTCGACATGAAAGGTGAATATGCAGAACATC	CCATGGTACCGGATCCAAAAGGCGGTGGTGGTGG
PcAHL17-RT-qPCR	CAAACCCAAACCACCCGTTAT	TGTTGCCGATGGCTGACG

图1 灰杨PcAHL17蛋白序列分析A：灰杨PcAHL17氨基酸序列与其他物种AHL17多重序列比对；B：系统进化树构建；Pc：灰杨；Pa：银白杨；Pt：毛果杨；Pn：黑杨；Ss：簸箕柳；Sp：红皮柳；Me：木薯；Gm：大豆；At：拟南芥；Nt：烟草；Gh：棉花；Hb：橡胶；Tc：可可；Cs：亚麻荠

Fig. 1 Sequence analysis of PcAHL17 protein in Populus × canescensA: Multiple sequence alignment of AHL17 amino acid sequences between Populus×canescens and other species. B: Construction of evolutionary tree. Pc: Populus × canescens;Pa: Populus alba; Pt: Populus trichocarpa; Pn: Populus nigra; Ss: Salix suchowensis; Sp: Salix purpurea; Me: Manihot esculenta; Gm: Glycine max; At: Arabidopsis thaliana; Nt: Nicotiana tabacum; Gh: Gossypium hirsutum; Hb: Hevea brasiliensis; Tc: Theobroma cacao; Cs: Camelina sativa

图2 镉胁迫下灰杨根茎叶中PcAHL17基因表达量的变化数值为3次重复实验结果的均值，以误差线表征平均标准差。不同小写字母表示差异显著（P<0.05），下同

Fig. 2 Changes of PcAHL17 gene expression in the roots, stems and leaves of Populus × canescens under cadmium stressThe data are the means of three repeated experimental results, with error bars indicating the standard deviation. Different lowercase letters denote significant differences (P<0.05), the same below

图3 灰杨PcAHL17转化拟南芥的DNA鉴定和荧光定量PCR鉴定A：T2代株系DNA检测；B：PcAHL17基因在野生型、转空载体、过表达拟南芥中的表达量，AtACTIN2为内参基因

Fig. 3 DNA identification and fluorescence quantitative PCR identification of A. thaliana transformed by PcAHL17 in Populus×canescensA: DNA detection of T2 genaration. B: Expression of PcAHL17 gene in wild-type, vector control, overexpressing A. thaliana, AtACTIN2 was used as internal control gene

图4 PcAHL17的亚细胞定位分析PcAHL17-GFP与PYR1-mCherry共定位。标尺=10 μm

Fig. 4 Subcellular localization analysis of PcAHL17PcAHL17-GFP co-localized with the PYR1-mCherry. Scale bar =10 μm

图5 拟南芥在各浓度CdCl2培养基上的萌发率A：CdCl2对幼苗生长的影响；B：幼苗萌发率统计分析

Fig. 5 Germination rates of A. thaliana on the media containing different concentrations of CdCl2A: Effects of CdCl2 on seed germination. B: Statistical analysis of the seed germination rate

图6 CdCl2对拟南芥根长生长的影响A：CdCl2对根长生长的影响；B：根系生长分析，白色线条表示拟南芥根的生长位置

Fig. 6 Effects of CdCl2 on the growth of root length of A.thalianaA: Effects of CdCl2 on the growth of root length. B: Analysis of root growth. The white lines indicate the growth position of Arabidopsis roots

图7 拟南芥在镉处理后的生长状态

Fig. 7 Growth state of A. thaliana after cadmium treatment

图8 CdCl2胁迫对不同拟南芥株系相对电导率的影响

Fig. 8 Effects of CdCl2 treatment on the relative electrical conductivity of different A. thaliana lines

图9 镉处理后根尖细胞H2O2荧光强度的测定

Fig. 9 Fluorescence intensity of H2O2 in the root-tip cells of A. thaliana after CdCl2 treatment

图10 拟南芥在镉胁迫下的Cd2+离子含量分析A：拟南芥地上部Cd2+离子含量；B：拟南芥地下部Cd2+离子含量；C：拟南芥根尖稳态Cd2+离子流；D：镉处理后根尖细胞Cd2+荧光强度

Fig. 10 Analysis of Cd2+ content in A. thaliana under cadmium stressA: Cd2+ content in aboveground parts of A. thaliana.B:Cd2+ content in underground parts of A. thaliana.C:Steady Cd2+ flux kinetics in A. thaliana root tips. D: The fluorescence intensity of Cd2+ in the root-tip cells of A. thaliana under CdCl2 treatment

图11 镉胁迫下各株系拟南芥中抗氧化酶基因表达量及蛋白酶活性检测

Fig. 11 Expressions of antioxidant enzyme gene and detection of protein activity in different A. thaliana lines after CdCl2 treatment

图12 镉胁迫下拟南芥各株系的叶绿素荧光参数

Fig. 12 Chlorophyll fluorescence parameters in A. thaliana lines under CdCl2 treatment

图13 镉处理后拟南芥各株系叶绿素相对含量

Fig. 13 Chlorophyll content in A.thaliana under CdCl2 treatment

图14 镉处理后拟南芥各株系的光合参数变化

Fig. 14 Photosynthetic parameters of A.thaliana under CdCl2 treatment

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