陆地棉基因GhWRKY40-1的克隆及表达分析

Cloning and Expression Analysis of the GhWRKY40-1 Gene in Gossypium hirsutum L.

【目的】WRKY转录因子通过激活下游一系列抗逆应答基因的表达而提高植株的综合抗性。本研究旨在研究WRKY转录因子在陆地棉中的功能。【方法】通过基因芯片筛选鉴定出1个逆境诱导的陆地棉WRKY转录因子基因,由于此基因编码蛋白含有1个典型的WRKY结构域,且与拟南芥At WRKY40具有较高相似性,故命名为GhWRKY40-1。采用电子克隆及反转录-聚合酶链反应技术获得GhWRKY40-1基因c DNA全长,并对其进行序列分析及结构与功能预测。【结果】GhWRKY40-1的开放阅读框为981 bp,编码326个氨基酸,预测蛋白相对分子质量为35.78×103,等电点为8.39。GhWRKY40-1在拟南芥原生质体中瞬时表达定位于细胞核,且具有转录激活活性。RT-PCR结果表明,GhWRKY40-1受甘露醇诱导上调表达。【结论】推测GhWYKY40-1可能参与干旱胁迫应答反应,并且在植物逆境胁迫中发挥重要作用。上述分析为进一步从分子水平上验证其抗逆生物学功能以及揭示棉花非生物胁迫抗逆机制奠定了基础。

[Objective] WRKY transcription factors enhance plant resistance by activating the expression of a series of stress-re- sponsive downstream genes. The purpose of our research was to analyze functions of WRK~ transcription factors in upland cot- ton. [Method] A stress-induced upland-cotton WRKY transcription factor gene was screened by cDNA microarray analysis. The gene, which was found to contain a typical WRKY domain and to share a highly similar amino acid sequence with AtWRKY40, was accordingly named GhWRKY40-1. The full-length cDNA of GhWRKY40-1 was obtained by electronic cloning and reverse transcription-polymerase chain reaction （RT-PCR） technology, with protein sequence analysis and structure-function prediction performed using bioinformatics software. [Result] According to our results, GhWRKY40-1 contains a 981-bp open reading frame encoding 326 amino acid residues, with a predicted relative molecular mass of 35.78 kDa and an isoelectric point of 8.39. In Arabidopsis protoplasts, GhWRKY40-1 was localized in the nucleusand was found to have transcriptional activation activity. RT-PCR analysis showed that GhWRKY40-1 was up-regulated under mannitol stress. [Conclusion] We hypothesize that Gh- WYKY40-1 is involved in drought stress response and plays an important role in plant stress responses. Our characterization and bioinformatics analyses have laid the foundation for future confirmation of the biological functions of resistance at the molecular level and elucidation of mechanisms of abiotic stress resistance in cotton.