《植物生理学报》 2014, 50(10): 1501-1509

胡杨PeECT8基因的克隆及功能分析

张洲嘉, 帅鹏, 苏艳艳, 尹伟伦, 夏新莉*

北京林业大学生物科学与技术学院, 林木育种国家工程实验室, 北京100083

通信作者：夏新莉；E-mail: xiaxl@bjfu.edu.cn；Tel: 010-62336400

摘　要：

ECT基因家族已在拟南芥中被发现并报道, 然而它们是否参与植物在逆境胁迫下的响应过程却鲜有报道。为研究胡杨PeECT8基因的功能, 从胡杨叶片cDNA中克隆出PeECT8基因, 构建CaMV 35S::PeECT8植物表达载体, 利用花序浸染法转化拟南芥, 经GUS组织化学染色和PCR检测, 获得转基因植株, 进而测定不同浓度盐(NaCl)和甘露醇胁迫下转基因拟南芥种子的萌发率、生长势和根长。测序结果表明, 该基因编码区长度为1 821 bp, 可编码606个氨基酸。蛋白序列比对发现, PeECT8基因与毛果杨PtrECT8同源基因所编码的氨基酸一致性达93.42%, PeECT8蛋白包含一个YT521-B-like保守结构域。相比于野生型, 过量表达PeECT8的拟南芥在不同浓度NaCl胁迫下萌发率降低, 在D-甘露醇胁迫下萌发率没有明显变化。在NaCl和D-甘露醇胁迫下, 转基因拟南芥根的伸长长度小于野生型。这表明PeECT8基因在盐胁迫和渗透胁迫下发挥负调控的作用。

关键词：胡杨; PeECT8; 胁迫; 转基因

收稿：2014-05-17   修定：2014-08-25

资助：国家自然科学基金(31070597和31270656)。

Cloning and Functional Analysis of PeECT8 in Populus euphratica

ZHANG Zhou-Jia, SHUAI Peng, SU Yan-Yan, YIN Wei-Lun, XIA Xin-Li*

College of Biological Sciences and Technology, National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing 100083, China

Corresponding author: XIA Xin-Li; E-mail: xiaxl@bjfu.edu.cn; Tel: 010-62336400

Abstract:

ECT gene family were found in Arabidopsis, but it is unknown whether they were involved in the pathway of the plant in response to stress or not. In order to study the function of PeECT8 homologous gene in poplar, the PeECT8 gene was cloned by RT-PCR from Populus euphratica. Sequencing results indicated that the length of PeECT8 is 1 821 bp encoding 606 amino acids. Blast analysis showed that PeECT8 shares over more than 93% homology in amino acid sequence with PtrECT8 homologue. The deduced amino acid sequence contained one conserved domain (YT521-B-like). Based on sequence analysis, the PeECT8 was fused to CaMV 35S promoter to characterize gene function. Transgenic Arabidopsis was obtained by floral dip method and analyzed by GUS detection and PCR. The results showed that the germination percentage of transgenic Arabidopsis was reduced under NaCl stress and was not affected by D-mannitol; the root length of transgenic Arabidopsis was shorter than that of wild type under NaCl and D-mannitol stresses. It indicates that the PeECT8 gene plays a negative role in response to stress condition.

Key words: Populus euphratica; PeECT8; stress; transgenic