巴西橡胶树HbWRKY1基因的克隆及转化烟草的初步研究

Cloning of HbWRKY1 Gene from Hevea brasiliensis and Its Transformation in Nicotiana tabacum

摘要:

利用RACE 结合RT-PCR 技术,从巴西橡胶树(Hevea brasiliensis)总RNA 中扩增得到长度为1234 bp 的WRKY 基因cDNA 全长编码序列。通过氨基酸同源性比对,该序列推导的氨基酸序列与蓖麻、白杨的WRKY 同源性分别为79% 和73%,表明分离的cDNA 序列为橡胶树WRKY 基因,命名为HbWRKY1。通过构建pCAMBIA1304-HbWRKY1 植物表达载体,经农杆菌GV3101 介导,将HbWRKY1 基因导入烟草(Nicotiana tabacum)中,对所获得的潮霉素抗性烟草株系进行PCR 鉴定。结果表明, HbWRKY1 基因已整合到65 株转基因植株中。干旱胁迫试验表明,HbWRKY1 的过量表达可以明显提高转基因烟草对干旱胁迫的耐受能力。这说明WRKY 基因与橡胶树抗旱能力之间存在一定的关系。

Abstract:

Using RACE and RT-PCR techniques, a novel member of WRKY gene family, named as HbWRKY1, was isolated from H. brasiliensis. Bioinformatics analysis showed that the full length cDNA of HbWRKY1 was 1234 bp, containing a 912 bp open reading frame and encoding 303 amino acids. The deduced amino acid sequence was highly homologous to WRKY proteins from Ricinus communis and Populus tremula × P. alba by 79% and 73%, respectively. The HbWRKY1 was constructed into pCAMBIA1304 expression vector, and transformed into tobacco (Nicotiana tabacum) mediated by Agrobacterium. Sixty-five hygromycin B (Hyg) resistant regenerated plantlets of tobacco were selected. PCR detection indicated that all the Hyg resistant tobacco plants contained the alien HbWRKY1 gene. It confirmed that the over-expression of HbWRKY1 under drought stress could obviously enhanced drought-tolerance of transgenic tobacco plantlets. Therefroe, it suggested that there were relationship between WRKY gene and drought tolerance of Hevea brasiliensis.