《植物生理学报》 2014, 50(4): 549-557

利用SSH文库技术分离鉴定常春藤应答甲醛胁迫相关基因

曾智东, 游览, 赵艳, 戚传娇, 李昆志, 陈丽梅*

昆明理工大学生命科学与技术学院生物工程技术研究中心, 昆明650500

通信作者：陈丽梅；E-mail: chenlimeikm@126.com；Tel: 0871-65920213

摘　要：

很多研究结果证实常春藤可以有效吸收气体甲醛, 本研究结果表明常春藤叶片也可以有效吸收液体甲醛, 甲醛吸收量和时间呈指数函数关系。2、4、6 mmol·L^-1液体甲醛处理均在常春藤叶片内诱发氧化胁迫, 但2 mmol·L^-1液体甲醛胁迫在常春藤叶片内诱发的氧化胁迫水平较低。此外, 2 mmol·L^-1液体甲醛胁迫还显著提高了常春藤叶片内可溶性糖和可溶性蛋白的含量, 说明常春藤对2 mmol·L^-1液体甲醛胁迫的抗性较强。通过构建2 mmol·L^-1液体甲醛胁迫2~48 h常春藤叶片的正向SSH cDNA文库, 分离鉴定常春藤叶片中的甲醛胁迫应答基因并对甲醛胁迫应答基因进行功能聚类, 结果说明光合作用和代谢相关基因占的比例最大, 表达分析结果证实光合作用和代谢相关基因在2 mmol·L^-1液体甲醛胁迫的不同阶段被诱导上调表达, 这些基因的上调表达可能和甲醛在常春藤叶片内的代谢脱毒有关。此外, 参与植物对多种生物和非生物胁迫应答的14-3-3蛋白基因(14-3-3p)也受2 mmol·L^-1液体甲醛胁迫的强烈诱导, 该基因的上调表达可能参与甲醛胁迫下常春藤叶片内可溶性蛋白的合成与抗氧化系统活性的调控作用, 是常春藤叶片应答甲醛胁迫的重要基因之一。

关键词：常春藤; 甲醛胁迫应答基因; SSH cDNA文库; 光合作用相关基因; 14-3-3p基因

收稿：2014-01-26   修定：2014-02-25

资助：国家自然科学基金(30970263)。

Isolation and Identification of Formaldehyde-Response Genes from Ivy (Hedera nepalensis var. sinensis) Leaves Using SSH cDNA Library

ZENG Zhi-Dong, YOU Lan, ZHAO Yan, QI Chuan-Jiao, LI Kun-Zhi, CHEN Li-Mei*

Biotechnology Research Center, College of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China

Corresponding author: CHEN Li-Mei; E-mail: chenlimeikm@126.com; Tel: 0871-65920213

Abstract:

Many studies have confirmed that ivy (Hedera nepalensis var. sinensis) can absorb gaseous formaldehyde (HCHO). The results of this study showed that ivy leaves could effectively absorb the liquid HCHO. The relationship between HCHO absorption and time fits the exponential function. Treatments of 2, 4, 6 mmol·L-1 liquid HCHO all induced oxidative stress in ivy leaves, however, the oxidative stress level in 2 mmol·L-1 HCHO-stress leaves was relative lower. Moreover, 2 mmol·L-1 liquid HCHO-stress significantly increased the content of soluble sugar and soluble protein in ivy leaves, indicating that ivy leaves was more resistant to 2 mmol·L-1 HCHO-stress. By constructing a suppression subtractive hybridization (SSH) cDNA library using ivy leaves treated with 2 mmol·L-1 liquid HCHO for 2–48 h, the HCHO-responsive genes in ivy leaves were isolated and identified. The function clustering for HCHO-stress responsive genes indicated that photosynthesis- and metabolism-related genes accounted for the two largest proportions. RT-PCR analysis confirmed that the expression of photosynthesis- and metabolism-related genes was induced in different stages of the 2 mmol·L-1 liquid HCHO-stress period. Up-regulated expression of these genes might be involved in HCHO-metabolism and -detoxification in ivy leaves. Moreover, a 14-3-3p gene, which is involved in responses to many biotic and abiotic stresses, was strongly induced by 2 mmol·L-1 liquid HCHO-stress. The up-regulated expression of 14-3-3p gene might be associated with the synthesis of soluble proteins and the regulation of antioxidant system activity in ivy leaves under HCHO stress. These results suggested that 14-3-3p gene might be one of the important HCHO stress-response genes.

Key words: Hedera nepalensis var. sinensis; formaldehyde-stress responsive genes; suppression subtractive hybridization (SSH) cDNA library; photosynthesis-related genes; 14-3-3p gene