东南景天SaWRKY7基因对镉胁迫的响应研究

Research on the response of SaWRKY7 gene to cadmium stress in Sedum alfredii Hance

【目的】镉对植物具有高毒性,WRKY转录因子广泛参与植物逆境胁迫。研究镉诱导下WRKY基因的胁迫响应和抗性机制。【方法】以超积累型东南景天为试材,通过PCR克隆得到1个东南景天WRKY转录因子家族基因序列,结合生物信息学方法,分析其蛋白质结构和亲缘关系,构建GFP(绿色荧光蛋白)融合蛋白,观察其亚细胞定位,利用酵母验证其转录激活活性,通过qRT-PCR分析在镉胁迫下该基因的表达特性和组织特异性。【结果】获得了1条新的东南景天WRKY转录因子家族基因并命名为SaWRKY7,包含完整的开放阅读框,长度为1 011 bp,共编码336个氨基酸,为WRKY基因家族Ⅱd成员。SaWRKY7定位在细胞核,属于核蛋白,不具有转录自激活活性。SaWRKY7在东南景天的根、茎、叶中均有表达,且在根中相对有较高的表达量。与对照相比,在100μmol/L CdCl_2处理0.5 h时,SaWRKY7在根茎叶中有较高的表达量,随后逐渐降低,属于早期诱导表达基因。【结论】SaWRKY7为WRKY转录因子家族成员,该基因定位在细胞核,不具有转录自激活活性,在早期响应镉胁迫诱导表达,推测其可能在东南景天镉积累或镉耐受过程中发挥重要作用。

【Objective】 Cadmium is highly toxic to plants. WRKY transcription factors are widely involved in stress response, therefor we studied the stress response and resistance mechanism of cadmium induced WRKY gene.【Method】 Gene sequences were obtained by PCR from the cDNA library of Sedum alfredii. Bioinformatics was used to analyze the protein structure.The green fluorescent protein fusion protein was constructed for the subcellular localization analysis. The transcriptional activation activity was verified with yeast. The expression characteristics and tissue specificity of the gene were analyzed by qRT-PCR under cadmium stress.【Result】 A new member of the family of WRKY transcription factor genes was obtained and named SaWRKY7. It contained a complete open reading frame, which was 1 011 base pairs in length, encoded 336 amino acids, and was a member of the Ⅱd family of WRKY genes. SaWRKY7 was found in the nucleus, is a nucleoprotein, and showed no transcriptional activation activity. SaWRKY7 was expressed in roots, stems and leaves of S. alfredii, with relatively high expression found in roots. SaWRKY7 showed a higher expression level in the rhizomes and leaves in the 0.5 h with 100 μmol/L cadmium treatment than that of the control, and then decreased gradually;thus, it belonged to the early induced expression gene.【Conclusion】 SaWRKY7 is a member of the family of WRKY transcription factor genes, which is found in the nucleus and does not have transcriptional self-activation activity. SaWRKY7 gene expression was induced by cadmium stress during the early stages. Results showed that SaWRKY7 might play an important role in S. alfredii response to cadmium stress.