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    山新杨PdaGLK1基因在盐胁迫响应中的功能研究

    Functional study of PdaGLK1 gene in salt stress response of Populus davidiana × Populus alba var. pyramidalis

    • 摘要:
      目的 G2-like(Golden 2-Like/GLK)转录因子,是MYB类转录因子中GARP超家族的重要成员,在植物生长发育过程中发挥关键作用。本研究通过探究PdaGLK1基因在山新杨盐胁迫响应过程中的功能,旨在为揭示山新杨的耐盐分子调控机制奠定理论基础,为林木抗逆分子育种提供关键的候选基因资源。
      方法 通过农杆菌介导的叶盘法获得PdaGLK1基因稳定转化的山新杨过表达和抑制表达株系。在NaCl胁迫条件下,观察各株系表型的变化,并对各株系进行组织化学染色和抗逆生理指标的测定,从表型、活性氧清除能力和细胞完整性方面探究PdaGLK1基因在山新杨响应盐胁迫过程中的功能。
      结果 (1)经抗性筛选和PCR鉴定获得2个PdaGLK1基因过表达株系(OE1、OE2)和2个PdaGLK1基因抑制表达株系(IE1、IE2)。qRT-PCR结果显示,OE1、OE2基因表达量分别为野生型的25.16和24.79倍,IE1、IE2基因表达量为野生型的 12.85%和11.92%,可用于后续实验。(2)NaCl胁迫处理后,PdaGLK1抑制表达株系的生长受到了严重的抑制作用,叶片变黄、枯萎、脱落,且鲜质量的下降程度明显大于野生型;而过表达株系未受到明显的生长抑制作用,鲜质量的下降程度低于野生型和抑制表达株系,表明PdaGLK1基因的表达可以增强山新杨对盐胁迫的耐受性。(3)盐胁迫下,过表达PdaGLK1株系中活性氧积累较少,抗氧化酶的活性高于野生型和抑制表达株系,丙二醛含量和相对电导率均低于野生型和抑制表达株系,细胞损伤程度减轻。
      结论 PdaGLK1基因能够通过提高体内活性氧清除能力,减少细胞损伤,保持细胞完整性,进而提高山新杨对盐胁迫的适应能力。本研究不仅为解析山新杨的PdaGLK1基因功能提供了重要依据,并为山新杨耐盐种质资源的创新与改良提供了关键的候选基因资源。

       

      Abstract:
      Objective G2-like (Golden 2-Like/GLK) transcription factors, as important members of GARP superfamily in MYB-like transcription factors, play a key role in plant growth and development. This study aims to explore the function of PdaGLK1 gene in salt stress response of Populus davidiana × Populus alba var. pyramidalis, so as to lay a theoretical foundation for revealing the molecular regulatory mechanism of salt tolerance in Populus davidiana × P. alba var. pyramidalis and provide key candidate gene resources for stress-resistant molecular breeding of forest trees.
      Method Stably transformed overexpression and repression expression plants of Populus davidiana × P. alba var. pyramidalis with the PdaGLK1 gene were obtained by Agrobacterium-mediated leaf disc method. Under NaCl stress conditions, the phenotypic changes of each line were observed, and histochemical staining and determination of stress-resistant physiological indexes were carried out for each line to explore the function of PdaGLK1 gene in salt stress response of Populus davidiana × P. alba var. pyramidalis from the aspects of phenotype, reactive oxygen species (ROS) scavenging capacity, and cell integrity.
      Result (1) Two overexpression lines (OE1, OE2) and two repression expression lines (IE1, IE2) of PdaGLK1 gene were obtained through resistance screening and PCR identification. The qRT-PCR results showed that gene expression levels of OE1, OE2 were 25.16 and 24.79 times of wild type, respectively, and the gene expression levels of IE1, IE2 were 12.85% and 11.92% of wild type. (2) After NaCl stress treatment, the growth of Populus davidiana × P. alba var. pyramidalis with repressed expression of PdaGLK1 was severely inhibited, with yellowing, wilting, and shedding of leaves, and the decrease in fresh mass was significantly greater than wild type; while the overexpressed Populus davidiana × P. alba var. pyramidalis was not significantly inhibited in growth, and the decrease in fresh mass was lower than wild type and repression expression plants, indicating that the expression of PdaGLK1 gene can enhance salt stress tolerance of Populus davidiana × P. alba var. pyramidalis. (3) Under salt stress, the overexpressed Populus davidiana × P. alba var. pyramidalis plants with PdaGLK1 gene had less ROS accumulation, higher antioxidant enzyme activities than the wild type and repression expression plants, and lower malondialdehyde (MDA) content and relative electrical conductivity than wild type and repression expression plants, with reduced cell damage.
      Conclusion The PdaGLK1 gene can improve salt stress adaptability of Populus davidiana × P. alba var. pyramidalis by enhancing in vivo ROS scavenging capacity, reducing cell damage, and maintaining cell integrity. This study not only provides an important basis for analyzing the function of PdaGLK1 gene in Populus davidiana × P. alba var. pyramidalis, but also provides key candidate gene resources for the innovation and improvement of salt-tolerant germplasm resources of Populus davidiana × P. alba var. pyramidalis.

       

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