棉花GhPIF4调控高温下花药败育机制初探

Mechanism of GhPIF4 regulating anther abortion under high temperature stress in cotton

PIF4属于PIF家族(PHYTOCHROME INTERACTING FACTORS FAMILY),是一个响应温度变化的关键性转录因子,广泛参与植物热信号传导和激素信号通路。本研究从陆地棉‘YZ1’中克隆获得GhPIF4基因,亚细胞定位结果表明,GhPIF4是一个核蛋白。qRT-PCR分析和proGhPIF4:GUS转基因棉花GUS染色结果显示,GhPIF4基因在棉花花药中高量表达,且其表达在高温敏感型材料‘H05’中受到高温的显著诱导。在棉花中超量表达GhPIF4,不同的转基因株系表现不同的育性,表达量高的3个转基因系(OE5、OE7和OE19)开花当天的花药不开裂,花粉的活力均低于对照‘YZ1’,而超表达程度略低的OE10,花药正常开裂,花粉活性正常,表明GhPIF4导致的花药败育具有剂量效应。对OE7和OE19在9~14 mm(绒毡层降解期)、14~19 mm(有丝分裂I期)中生长素的含量以及生长素合成关键基因GhTAA1、GhYUC2、GhCYP71A13的表达分析发现基因的表达与生长素含量下降变化一致。推测超表达GhPIF4在花药后期可能部分模拟棉花的高温响应状态,而组成型超表达GhPIF4可能改变了营养器官或早期花药中生长素的含量,亦或花药中生长素含量过低也会导致花药败育。以上结果为深入解析GhPIF4基因功能及了解高温胁迫下棉花花药败育的机制提供参考。

PIF4,which belongs to the phytochrome interacting factor family,is a key transcription factor in response to temperature changes,and is widely involved in plant heat signaling transduction and hormonal signaling pathways.In this study,the GhPIF4 gene was cloned from‘YZ1’(Gossypium hirsutum).Subcellular localization results indicated that GhPIF4 is a nuclear located protein.qRT-PCR analysis and GUS staining of proGhPIF4:GUS transgenic cotton plants showed that the GhPIF4 gene was highly expressed in the anther,and its expression was significantly induced by high temperature(HT)in the‘H05’(HT sensitive line).With the overexpression of GhPIF4,different cotton transgenic lines showed different male fertility performances.Three transgenic lines with high GhPIF4 expression,OE5,OE7,and OE19,showed indehiscent anthers in the flower on the day of flowering and lower pollen activity than wild type‘YZ1’.However,the transgenic line OE10 with slighter overexpression showed normal anther dehiscence and pollen activity.These results indicated that GhPIF4 causes anther abortion in an expression level-dependent manner.Furthermore,the IAA content was measured in OE7 and OE19 anthers at the tapetum degradation stage(9–14 mm buds)and mitotic stage(14–19 mm buds).As a result,the IAA content in OE7 and OE19 decreased compared to that in‘YZ1’.Then,we analyzed the expression of auxin biosynthesis genes,including GhTAA1,GhYUC2,and GhCYP71A13,showing that the expression of these genes was down-regulated in OE7 and OE19,which was consistent with the decrease in IAA content.We hypothesize that overexpression of GhPIF4 might partially simulate the response of late stage anthers to HT,while constitutive overexpression of GhPIF4 may alter the auxin content in vegetative organs or early stage anthers,or the decline of IAA may lead to the anther abortion.These results provide a reference for further analysis of the function of GhPIF4 gene and understanding the mechanism of cotton anther abortion under HT.