海岛棉GbWRKY40基因的克隆及特征分析

Cloning and Characterization of the GbWRKY40 Transcription Factor Gene from Gossypium barbadense L.

【目的】WRKY转录因子调控多种生物学进程,包括植物生长发育和应答多种环境胁迫。本研究旨在分析WRKY转录因子在海岛棉纤维发育中的功能。【方法】从海岛棉中克隆了1个WRKY转录因子基因GbWRKY40,进行同源性分析、多序列比对,利用荧光定量聚合酶链式反应分析其表达模式,通过构建酵母表达载体并转化酵母菌株AH109研究其转录激活活性。【结果】该基因c DNA全长1713 bp,5'非编码区长261 bp,3'非编码区长510 bp;开放阅读框长942 bp,编码313个氨基酸,预测相对分子质量约为34.138×103,等电点为8.46,包含5个外显子和4个内含子;其编码蛋白含有1个WRKY保守区(WRKYGQK)和1个锌指基序(C-X5-C-X23-H-X1-H),属于WRKY家族第Ⅱ类a组,包含3个核定位信号区,与陆地棉GhWRKY40同源性最高。GbWRKY40在根和开花后25 d纤维中表达量高,而且不具有转录激活活性。【结论】GbWRKY40可能参与调控棉纤维次生壁发育。

[Objective] The WRKY transcription factors modulate various biological processes, including plant growth, development, and responses to various environmental stresses. This study aimed to clarify the role of WRKY transcription factors related to cotton fiber development. [Method] The GbWRKY40 gene, which encodes a WRKY transcription factor, was isolated from cotton （Gossypium barbadense L.）. The obtained sequence was analyzed for homology with genes from other cotton species using the DNAMAN7 program. The sequences of the homologous genes were aligned with Clustalx1.83. Additionally, the GbWRKY40 expression pattern was analyzed by a quantitative real-time polymerase chain reaction. Furthermore, a yeast expression vector was constructed and inserted into yeast strain AH109 cells to investigate the transcriptional activity of GbWRKY40. [Result] Full-length GbWRKY40 cDNA contains 1713 nucleotides, including a 942 bp open reading frame, a 261 bp 5＇-untranslated region, and a 510 bp 3＇-untranslated region. The open reading frame was predicted to encode a 313 amino acid protein with a relative molecular mass of 34.138 × 103 and an isoelectric point of 8.46. The genomic GbWRKY40 sequence was observed to comprise five exons and four introns. The predicted GbWRKY40 protein includes a WRKY domain consisting of approximately 60 amino acids, including the conserved WRKYGQK sequence and a zinc-finger motif （C-X5-C-X23-H-X1-H）. These characteristics indicated that GbWRKY40 belongs to group IIa of the WRKY family. Moreover, GbWRKY40 contains three putative nuclear localization signals. GbWRKY40 is highly homologous to GhWRKY40. Expression analyses involving a quantitative real-time polymerase chain reaction revealed that GbWRKY40 is highly expressed in cotton roots and fibers at 25 days post anthesis. Transcriptional activation assay results suggested that the function of the GbWRKY40 transcription factor is unrelated to the activation of transcription. [Conclusion] These results imply that GbWRKY40 may be involved in regulating secondary cell wal development in cotton fibers.