毛白杨 PtoGRF9 基因的克隆及其调控杨树耐旱性的功能解析

Cloning of PtoGRF9 from Populus tomentosa and functional characterization of its role in drought tolerance

摘要

【目的】GRF 转录因子在植物发育与胁迫应答中功能保守，但其在杨树耐旱性中的分子作用尚未见系统报道。本研究解析毛白杨 PtoGRF9 的生物学功能，为阐释杨树“生长-抗逆”平衡机制及耐旱分子育种提供理论依据与基因资源。【方法】从毛白杨中克隆 PtoGRF9，进行系统进化、蛋白结构、启动子及表达模式分析；利用农杆菌介导的叶盘转化法创制过表达（ PtoGRF9-OE）与沉默（ PtoGRF9-RNAi）84K 杨树株系，测定干旱胁迫下光合生理指标及抗氧化酶活性；结合 DNA 亲和纯化测序（DAP-seq）数据筛选并初步验证下游靶基因。【结果】（1）PtoGRF9 编码 457 个氨基酸，包含 1 个 QLQ 结构域和 2 个 WRC 结构域。（2） PtoGRF9 启动子含有多个与植物生长发育、应激响应及生物与非生物胁迫相关的顺式作用元件。反转录荧光定量 PCR（RT-qPCR）分析表明 PtoGRF9 在发育叶片中表达量较高，且受干旱胁迫显著诱导。（3） PtoGRF9-OE 株系在短期干旱胁迫下的光合参数及过氧化物酶活性等生理指标显著高于 WT，而 PtoGRF9-RNAi 株系则表现相反。（4）DAP-seq 及 RT-qPCR 共同验证 PtoSHN1、 PtoERD7、 PtoERECTA 和 PtoPIP1 等参与蜡质合成和气孔发育的基因为 PtoGRF9 的潜在靶基因。【结论】 PtoGRF9 是正向调控杨树耐旱性的转录因子，在维持叶片发育与抗逆性平衡中发挥关键作用。研究结果不仅为理解杨树生长发育与抗逆的分子机制提供了新视角，也为耐旱分子育种鉴定了关键候选基因。

Abstract

[Objective] GRF transcription factors play conserved roles in plant development and stress responses, yet their molecular contributions to drought tolerance in poplar remain unclear. Here, we functionally characterize PtoGRF9 in Populus tomentosa, elucidate its role in the growth-defence balance and provide a target for molecular breeding of drought resistant poplar. [Method] We first cloned PtoGRF9 from Populus tomentosa, performed phylogenetic, structural, promoter and expression analyses; then, Agrobacterium-mediated leaf-disc transformation was employed to generate both over-expression (PtoGRF9-OE) and RNAi-silenced (PtoGRF9-RNAi) 84K poplar lines, which were then subjected to drought stress to evaluate photosynthetic performance and antioxidant enzyme activities. Finally, DNA affinity purification sequencing (DAP-seq) data were integrated to identify and preliminarily validate the downstream target genes of PtoGRF9. [Result] (1) PtoGRF9 encodes a protein of 457 amino acids that contains one QLQ domain and two WRC domains. (2) The PtoGRF9 promoter contains multiple cis-acting elements involved in plant growth and development, stress signaling, and biotic/abiotic stress responses. Reverse transcription-quantitative PCR (RT-qPCR) assays showed that PtoGRF9 has a relatively high expression level in developing leaves and is significantly induced by drought stress. (3) Under short-term drought, PtoGRF9-OE lines exhibited significantly higher photosynthetic performance and peroxidase activities than WT, whereas PtoGRF9-RNAi lines displayed the opposite trend. (4) DNA affinity purification sequencing (DAP-seq) and RT-qPCR analyses identified the wax-biosynthesis and stomatal-development genes SHN1, ERD7, ERECTA and PIP1 as potential PtoGRF9 targets. [Conclusion] PtoGRF9 is a transcription factor that positively regulates drought tolerance in poplar and plays a key role in maintaining the balance between leaf development and stress resistance. Our findings not only provide new insights into the molecular mechanisms underlying poplar growth, development, and stress tolerance but also identify a key candidate gene for drought-tolerant molecular breeding.

关键词

毛白杨 / PtoGRF9 / 耐旱性 / 叶片发育 / 转录因子

Key words

Populus tomentosa / PtoGRF9 / drought tolerance / leaf development / transcription factor

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基金资助

山东省自然科学基金青年科学基金项目(ZR2024QC117)