水母雪兔子通气组织形成相关基因SmPAD4的克隆及表达分析

韦鎔宜<sup>1; 2; 3</sup>; 段 鹏<sup>4</sup>; 李培兰<sup>1; 2; 3</sup>; 罗 丹<sup>1; 2; 3</sup>; 史国民<sup>2; 3</sup>; 代吴斌<sup>2; 3</sup>; 李凤珍<sup>1; 2; 3</sup>; 何 涛<sup>1; 2; 3*</sup>

引用本文:	韦鎔宜, 段鹏, 李培兰, 罗丹, 史国民, 代吴斌, 李凤珍, 何涛.水母雪兔子通气组织形成相关基因SmPAD4的克隆及表达分析[J].广西植物,2024,44(12):2265-2278.[点击复制]
	WEI Rongyi, DUAN Peng, LI Peilan, LUO Dan, SHI Guomin, DAI Wubin, LI Fengzhen, HE Tao.Cloning and expression of aerenchyma formation-related gene SmPAD4 in Saussurea medusa[J].Guihaia,2024,44(12):2265-2278.[点击复制]

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*水母雪兔子通气组织形成相关基因SmPAD4的克隆及表达分析*
韦鎔宜^1,2,3, 段鹏⁴, 李培兰^1,2,3, 罗丹^1,2,3, 史国民^2,3, 代吴斌^2,3, 李凤珍^1,2,3, 何涛^1,2,3*
1. 青海大学生态环境工程学院, 西宁 810016;2. 青海省园林植物与观赏园艺重点实验室, 西宁 810016;3. 青海大学省部共建三江源生态与高原农牧业国家重点实验室, 西宁 810016;4. 青海大学畜牧兽医科学院, 西宁 810016

摘要:

通气组织是水母雪兔子(Saussurea medusa)应对极端环境的适应性结构,其形成通常伴随着细胞程序性死亡(programmed cell death,PCD)的发生,而细胞的死亡与通气组织的形成通常受到PAD4基因(Phytoalexin Deficient 4)的调控,但PAD4如何调控水母雪兔子通气组织形成的机制尚不明确。该研究以水母雪兔子为试验材料,利用同源克隆和RACE技术克隆了通气组织形成相关基因SmPAD4,对其序列、系统进化、表达和亚细胞定位等进行分析,并采用hi-TAIL PCR技术扩增其启动子,以探讨该基因在环境适应中的功能。结果表明:(1)SmPAD4基因cDNA全长为2 047 bp(GenBank登录号为OR766038),包括1 866 bp的开放阅读框,编码621 个氨基酸,分子式为C₃₁₆₃H₄₉₀₆N₈₄₈O₉₁₀S₂₆,该蛋白为碱性亲水性不稳定蛋白。(2)系统进化树分析显示,SmPAD4与刺苞菜蓟CcPAD4的氨基酸序列相似度最高。(3)扩增出1 049 bp的SmPAD4启动子序列,包含有光响应元件、低氧应答元件、干旱和生长素应答元件等顺式作用元件。(4)实时荧光定量(qRT-PCR)分析显示,SmPAD4基因在根、茎和叶中均有表达且在叶中的表达量最高; 在紫外和低氧胁迫下SmPAD4基因在叶和茎中表达量均上调,根中表达量下调。(5)亚细胞定位显示,SmPAD4分布于细胞核、细胞膜和叶绿体。该研究表明,SmPAD4基因拥有独特的蛋白结构域,并且响应低氧和紫外两种环境胁迫,在通气组织的形成以及对逆境胁迫的响应中具有重要作用,为进一步探究SmPAD4基因在水母雪兔子适应环境过程中的作用提供了理论依据。

关键词: 水母雪兔子, SmPAD4, 通气组织, 表达分析, 低氧胁迫, 紫外胁迫

DOI：10.11931/guihaia.gxzw202312028

分类号:Q943

文章编号:1000-3142(2024)12-2265-14

基金项目:国家自然科学基金(31960222)。

Cloning and expression of aerenchyma formation-related gene SmPAD4 in Saussurea medusa

WEI Rongyi^1,2,3, DUAN Peng⁴, LI Peilan^1,2,3, LUO Dan^1,2,3, SHI Guomin^2,3, DAI Wubin^2,3, LI Fengzhen^1,2,3, HE Tao^1,2,3*

1. School of Ecol-Environmental Engineering, Qinghai University, Xining 810016, China;2. Key Laboratory of Landscape Plants of Qinghai Province, Xining 810016, China;3. State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University,Xining 810016, China;4. Academy of Animal Science and Veterinary, Qinghai University, Xining 810016, China 1. School of Ecol-Environmental Engineering, Qinghai University, Xining 810016, China; 2. Key Laboratory of Landscape Plants of Qinghai Province, Xining 810016, China; 3. State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University,Xining 810016, China; 4. Academy of Animal Science and Veterinary, Qinghai University, Xining 810016, China

Abstract:

The aerenchyma is an adaptive structure of Saussurea medusa in response to extreme environments, and its formation is usually accompanied by programmed cell death(PCD). The death of cells and the formation of aerenchyma are typically regulated by the PAD4 gene(Phytoalexin Deficient 4). However, the mechanism by which SmPAD4 regulates the formation of aerenchyma in S. medusa remains unclear. In this study, S. medusa was used as the experimental material, and the gene SmPAD4 related to aerenchyma formation was cloned by homologous cloning and RACE technology, and its sequence, phylogenetic evolution, expression and subcellular localization were analyzed, and its promoter was amplified by hi-TAIL PCR technology to explore its function in environmental adaptation. The results were as follows:(1)The cDNA of SmPAD4 gene was successfully cloned with a total length of 2 047 bp(GenBank accession number OR766038), including an open reading frame of 1 866 bp, encoding 621 amino acids, a molecular formula of C₃₁₆₃H₄₉₀₆N₈₄₈O₉₁₀S₂₆. The protein was an alkaline and hydrophilic unstable protein.(2)Phylogenetic tree analysis showed that SmPAD4 had high similarity with CcPAD4 of Cynara cardunculus.(3)A length of 1 049 bp promoter sequence of SmPAD4 was amplified, which included cis-acting elements such as light response element, hypoxia response element, dry and auxin response elements.(4)Real-time quantitative fluorescence(qRT-PCR)analysis showed that SmPAD4 gene was expressed in root, stem and leaf, and the expression level was the highest in leaf. Under ultraviolet and hypoxia stresses, the expression of SmPAD4 gene was up-regulated in leaf and stem, and down-regulated in root.(5)Subcellular localization showed that SmPAD4 was distributed in the nucleus, cell membrane, and chloroplast. The results show that SmPAD4 gene has a unique protein domain and it responds to hypoxia and ultraviolet environmental stresses, so it plays an important role in the formation of aerenchyma and the response to adversity stress. This study provides theoretical basis for further exploring the role of SmPAD4 gene in the environmental adaptation process of Saussurea medusa.

Key words: Saussurea medusa, SmPAD4, aerenchyma, expression analysis, hypoxia stress, ultraviolet stress