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短季棉(Gossypium hirsutum)的早熟往往伴随着早衰,挖掘和鉴定衰老相关基因对于选育早熟不早衰棉花新品种具有重要意义,而WRKY转录因子广泛参与调控植物衰老过程。本研究克隆了陆地棉WRKY转录因子Gh WRKY11(Gen Bank accession No.JN604988),c DNA全长1 055 bp,包含1 008 bp开放阅读框(open reading frame,ORF),编码335个氨基酸,属于WRKY转录因子Ⅱd亚组。蛋白序列系统进化分析表明,Gh WRKY11与拟南芥(Arabidopsis thaliana)At WRKY11的相似度较高。基因结构分析表明,Gh WRKY11基因有3个外显子和2个内含子。亚细胞定位结果表明,Gh WRKY11蛋白定位在洋葱(Allium cepa)表皮的细胞核。q RT-PCR结果分析表明,Gh WRKY11在盛花期的棉花叶片中优势表达,在子叶衰老过程中表达下调。赤霉素(gibberellin,GA3)处理棉花幼苗后,Gh WRKY11在2、4、8和12 h的表达显著(P<0.05)或极显著(P<0.01)上调;水杨酸(salicylic acid,SA)处理后,Gh WRKY11的表达量随处理时间的增加而逐渐升高,并且在2、4、8和12 h的表达极显著(P<0.01)上调;同时,Gh WRKY11可以在某些时间点被脱落酸(abscisic acid,ABA)、乙烯(ethylene,ET)、H2O2和茉莉酸(jasmonic acid,JA)诱导表达上调。Gh WRKY11在受到低温(12℃)胁迫处理时表达上调,且在2、8和12 h达极显著(P<0.01);盐胁迫(200 mmol/L Na Cl)处理后,Gh WRKY11在2、4、8和12 h的表达显著(P<0.05)或极显著(P<0.01)上调;15%聚乙二醇6000(polyethylene glycol 6000,PEG6000)处理后,在12 h的表达量极显著(P<0.01)上调;损伤处理对该基因的表达没有显著影响。播种45 d后,与野生型拟南芥相比,Gh WRKY11拟南芥过表达植株衰老延缓,由此推断,Gh WRKY11可以延缓植株衰老,初步鉴定Gh WRKY11是衰老的负调控因子。该基因的克隆和功能的初步分析为创制转基因抗早衰棉花材料提供基础资料。
The early maturity of Gossypium hirsutum is often associated with premature aging, and the identification and mining of genes related to senescence is of great importance for the breeding of new varieties of early maturity cotton. WRKY transcription factor is widely involved in the regulation of plant senescence. In this study, we cloned the WRKY transcription factor Gh WRKY11 (GenBank accession No.JN604988), which contains a full-length cDNA of 1 055 bp and contains an open reading frame (ORF) of 1 008 bp and encodes a protein of 335 amino acids. It belongs to WRKY Transcription factor IId subgroup. Phylogenetic analysis of the protein sequence showed that Gh WRKY11 is highly similar to At WRKY11 in Arabidopsis thaliana. Gene structure analysis showed that Gh WRKY11 gene has three exons and two introns. The results of subcellular localization showed that Gh WRKY11 protein localized in the nucleus of allium cepa epidermis. q RT-PCR results showed that Gh WRKY11 was predominantly expressed in full-flowering cotton leaves and down-regulated in cotyledon senescence. The expression of Gh WRKY11 at 2, 4, 8 and 12 h was significantly (P <0.05) or significantly higher (P <0.01) after treated with gibberellin (GA3) ), The expression of Gh WRKY11 increased gradually with the increase of treatment time, and it was significantly up-regulated (P <0.01) at 2, 4, 8 and 12 h. At the same time, Gh WRKY11 could up- It was induced by abscisic acid (ABA), ethylene (ET), H2O2 and jasmonic acid (JA). The expression of Gh WRKY11 was up-regulated at low temperature (12 ℃), and reached the peak at 2, 8 and 12 h (P <0.01). After treatment with 200 mmol / L NaCl, (P <0.05) or significantly (P <0.01) upregulation at 4, 8 and 12 h, and significant at 12 h after treatment with 15% polyethylene glycol 6000 (PEG 6000) P <0.01). The injury treatment had no significant effect on the gene expression. Compared with wild-type Arabidopsis thaliana, Gh WRKY11 overexpression delayed the plant growth 45 days after sowing, suggesting that Gh WRKY11 can delay plant senescence. Preliminary identification of Gh WRKY11 is a negative regulator of aging. Preliminary analysis of the cloning and function of this gene provided the basic information for the creation of transgenic cotton material with anti-premature aging.