PagAPY1基因调控银腺杨耐旱性的作用机制研究

王质璞; 李卓蓉; 罗志斌; 邓澍荣

doi:10.12302/j.issn.1000-2006.202309014

PagAPY1基因调控银腺杨耐旱性的作用机制研究

王质璞, 李卓蓉, 罗志斌, 邓澍荣

南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (6) : 105-112.

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南京林业大学学报（自然科学版） ›› 2023, Vol. 47 ›› Issue (6) : 105-112. DOI: 10.12302/j.issn.1000-2006.202309014

研究论文

PagAPY1基因调控银腺杨耐旱性的作用机制研究

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Mechanisms of PagAPY1 in regulating drought tolerance in Populus alba × P. glandulosa

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摘要

【目的】Apyrase是水解细胞内/外核苷磷酸的关键酶,在调控植物生长发育和逆境响应中起重要作用。本研究鉴定银腺杨(Populus alba × P. glandulosa)中PagAPY1的分子功能,揭示其促进耐旱性的作用机制。【方法】利用生物信息学方法鉴定银腺杨PagAPY1基因,构建PagAPY1-YFP融合蛋白,观察PagAPY1的亚细胞定位,利用农杆菌介导的转基因技术获得过表达PagAPY1转基因银腺杨,利用实时荧光定量PCR(qRT-PCR)检测野生型和转基因杨树中PagAPY1及其他抗逆基因表达,分析干旱胁迫下野生型和转基因杨树生长表型、根系发育和叶片保水性能等生理指标。【结果】PagAPY1具有Apyrase保守结构域,定位于高尔基体中,参与水解NTP/NDP。PagAPY1表达受干旱胁迫诱导。在干旱胁迫下,PagAPY1过表达杨树的存活率、根系生物量和不定根生长等相比野生型显著提高;同时,过表达杨树的气孔开度相比野生型降低。与野生型相比,过表达株系对ABA诱导的气孔关闭更为敏感。此外,过表达株系中活性氧信号和抗氧化酶相关基因在干旱胁迫下显著上调表达。【结论】干旱胁迫下,PagAPY1通过维持蛋白糖基化和胞外ATP(eATP)稳态平衡,促进生长素极性运输,提高根系生长,促进气孔闭合,减少叶片水分散失,同时激活活性氧信号,增加抗氧化酶系统活性,从而提高了杨树抗旱性。

Abstract

【Objective】 Apyrases are key enzymes that hydrolyze nucleoside di- and triphosphates inside or outside the cell, which play crucial roles in regulating plant growth and stress responses. This study identified the molecular function of Apyrase gene PagAPY1 in Populus alba × P. glandulosa and elucidated the regulatory mechanisms of PagAPY1 in enhancing drought tolerance in poplars. 【Method】 Using bioinformatics approaches, the PagAPY1 gene was identified in genome of P. alba × P. glandulosa. A PagAPY1-YFP fusion protein was constructed for subcellular localization analysis. PagAPY1-overexpressing transgenic poplars were obtained through agrobacterium-mediated transformation. qRT-PCR was used to detect the expression of PagAPY1 and other stress-related genes in both wild type and transgenic poplars. The growth phenotype, root development, and leaf water retention were analyzed under drought stress. Leave stomatal aperture was measured in both WT and transgenic poplars under drought or ABA treatment. 【Result】 The protein coded by PagAPY1 possessed conserved ACR domains and amino acid residues critical for enzyme catalytic activity. PagAPY1 was localized to the Golgi apparatus, where it could participate in the hydrolysis of NTP/NDP. The expression of PagAPY1 was induced by drought. Eight PagAPY1-overexpressing transgenic poplars were identified and three OE lines with the highest PagAPY1 expression levels were selected for further experiments. Under drought stress, the PagAPY1-OE transgenic lines showed improved survival rates, plant height, root length, root biomass compared with the wild-type poplars. Under osmotic stress, the transgenic plants exhibited greater adventitious root growth compared with the wild type. Meanwhile, the stomatal aperture was more significantly decreased in the transgenic lines during drought condition. In contrast to wild-type, the OE lines exhibited greater sensitivity to ABA-induced stomatal closure. The expression of genes involved in ROS signaling and genes coding antioxidant enzymes was significantly upregulated in the PagAPY1-overexpressing lines under drought condition. 【Conclusion】 Under drought stress, PagAPY1 facilitates auxin polar transport by maintaining protein glycosylation and eATP homeostasis, thereby enhances root growth in the overexpression poplars. Meanwhile, through regulating eATP levels in leaves, PagAPY1 promotes stomatal closure to reduce leaf water loss under drought stress. PagAPY1-overexpressing also increases the expression levels of genes that are involved in ROS signaling and antioxidant enzyme system, thereby enhancing drought resistance in poplars.

导出引用

王质璞, 李卓蓉, 罗志斌, 等. PagAPY1基因调控银腺杨耐旱性的作用机制研究[J]. 南京林业大学学报（自然科学版）. 2023, 47(6): 105-112 https://doi.org/10.12302/j.issn.1000-2006.202309014

WANG Zhipu, LI Zhuorong, LUO Zhibin, et al. Mechanisms of PagAPY1 in regulating drought tolerance in Populus alba × P. glandulosa[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2023, 47(6): 105-112 https://doi.org/10.12302/j.issn.1000-2006.202309014

中图分类号： S718;Q78

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