《植物生理学报》 2015, 51(5): 754-762

蝴蝶兰S-腺苷甲硫氨酸合成酶基因的克隆及低温下的表达分析

袁秀云¹, 雷志华², 梁芳¹, 蒋素华¹, 许申平¹, 王默霏¹, 崔波^1,*

郑州师范学院¹生物工程研究所, ²生命科学学院, 郑州450044

通信作者：崔波；E-mail: laocuibo@163.com；Tel: 0371-65501086

摘　要：

利用RT-PCR和RACE技术从蝴蝶兰品种‘满天红’中克隆获得SAMS基因的cDNA序列, GenBank登录号为KP966096。用生物信息学方法预测分析其序列, cDNA全长1 550 bp, 含有1个1 194 bp的完整开放阅读框(ORF), 编码397个氨基酸, 其氨基酸序列与多种植物的SAMS蛋白有很高的相似性。系统进化树分析显示, 蝴蝶兰SAMS与油棕的SAMS蛋白亲缘关系较近。采用GAPDH、ACTIN和EF1α这3个不同的内参基因作为标准对蝴蝶兰SAMS基因进行实时荧光定量分析, 结果表明, 在13 ℃/8 ℃的昼夜温度条件下, 蝴蝶兰SAMS基因的转录表达在3、6、9和15 d时明显升高, 恢复正常温度条件后, 该基因的表达下降; 在4 ℃低温条件下, SAMS基因的表达在处理0.5、1、2和4 h内明显升高, 处理8 h时其表达开始下降, 在处理12、24和48 h时, 其表达水平没有明显的升高和下降。结果表明该基因参与了蝴蝶兰低温胁迫的分子调控。

关键词：蝴蝶兰; S-腺苷甲硫氨酸合成酶; 低温胁迫; 基因克隆; 实时荧光定量PCR

收稿：2015-03-25   修定：2015-04-09

资助：河南省科技攻关项目(092102110128)。

Cloning of a S-Adenosylmethionine Synthetase Gene from Phalaenopsis amabilis and Its Expression under Low Temperature

YUAN Xiu-Yun¹, LEI Zhi-Hua², LIANG Fang¹, JIANG Su-Hua¹, XU Shen-Ping¹, WANG Mo-Fei¹, CUI Bo^1,*

¹Institute of Bioengineering, ²College of Life Science, Zhengzhou Normal University, Zhengzhou 450044, China

Corresponding author: CUI Bo; E-mail: laocuibo@163.com; Tel: 0371-65501086

Abstract:

The full-length sequence cDNA of SAMS (GenBank accession number: KP966096) was cloned from Phalaenopsis amabilis cultivar ‘Red Sky’ using RT-PCR combined with RACE techniques. This sequence consists of 1 550 bp with an intact open reading frame of 1 194 bp, encoding a polypeptide of 397 amino acids. Homology analysis showed that the deduced SAMS protein was highly homologous to SAMS proteins from different species. Phylogenetic analysis indicated that SAMS was closely related to the SAMS of Elaeis guineensis. The results of real-time fluorescent quantitative PCR using the combination of GAPDH, ACTIN and EF1α as internal control genes showed that the transcript levels of SAMS in P. amabilis were increased obviously under day/night 13 ℃/8 ℃ for 3, 6, 9 and 15 d, then declined after the recovery of normal temperature. Under the cold stress of 4 ℃, the expressions of SAMS were also increased significantly at 0.5, 1, 2 and 4 h after cold treatment, and declined at 8 h to the level before treatment. The transcript levels of SAMS were not obviously up-regulated and down-regulated at 12, 24 and 48 h after cold treatment. It suggested that SAMS play a role in cold tolerance in P. amabilis.

Key words: Phalaenopsis amabilis; S-adenosylmethionine synthetase; low temperature stress; gene cloning; real-time fluorescent quantitative PCR