转录因子TgWRKY24响应ABA调控天山郁金香种子休眠的分子机制

China is rich in tulip resources. It is an important way to breed new tulip cultivars by distant hybridization using native wild Tulipa resources. T. thianschanica is an excellent germplasm resource distributed in China with bright yellow flowers and strong resistance. However, its seeds have deep dormancy. Transcriptome sequencing showed that a transcription factor, TgWRKY24, played an important role in regulating seed dormancy in response to ABA treatment in tulip, but its molecular mechanism was unknown. This project intends to study the mechanism of TgWRKY24 in ABA signal transduction and regulation of seed dormancy. The full length of TgWRKY24 will be cloned and the gene structure and tissue specific expression will also be analyzed. The technologies of UPLC-MS/MS, qRT-PCR, Western bolting and VIGS will be used to investigate the expression of TgWRKY24 in response to ABA during seed dormancy. Transcriptome sequencing results will be used to forecast the other genes in the ABA signaling pathway related to the expression of TgWRKY24 and clone their promoters. The electrophoretic mobility shift assay and a yeast one-hybrid system will be used to screen and verify the downstream target genes of TgWRKY24. The results will be expected to explain the function of TgWRKY24 transcription factor in regulating tulip seed dormancy in response to ABA, and establish a new ABA signaling network for regulating seed dormancy in plant.

我国郁金香属植物资源丰富，充分利用我国原产的野生资源进行远缘杂交是选育郁金香品种的一条重要途径。天山郁金香是原产我国的优异种质资源，花鲜黄色，抗逆性强，但其种子休眠极深。通过转录组测序分析发现，转录因子TgWRKY24在天山郁金香响应ABA调控种子休眠中起重要作用，但其分子机制不详。本项目拟围绕这一机制开展研究：克隆TgWRKY24全长，分析基因结构并进行组织特异性表达分析；利用UPLC-MS/MS、qRT-PCR、Western bolting等技术，探明TgWRKY24在种子休眠过程中响应外源ABA的表达特性；预测与TgWRKY24表达相关的ABA信号通路中的其他基因，并进行启动子克隆，利用EMSA和酵母单杂交技术筛选和验证TgWRKY24的下游靶基因。研究结果可望解析TgWRKY24响应ABA调控郁金香种子休眠的功能，建立调控种子休眠的ABA信号网络新途径，丰富植物种子休眠调控机理。

郁金香种子普遍具有较深的休眠特性，低温和植物激素可以有效解除休眠。本研究前期发现原产我国的天山郁金香种子在低温层积条件下，转录因子TgWRKY24在响应ABA调控种子的休眠中具有重要作用。为此，本项目借助RACE-PCR技术克隆得到了TgWRKY24基因全长，利用生信工具对其系统进化与蛋白结构进行了分析，亚细胞定位该基因位于细胞核中，不同组织特异性表达显示该基因在鳞茎中的表达量最高，根中最低。通过测定不同浓度的外源植物激素ABA与FLU（ABA合成抑制剂）处理后种子内源ABA含量与TgWRKY24基因表达量，并与清水（CK）比较，发现种子在休眠解除过程中内源ABA含量与TgWRKY24基因表达量均呈现先升高后降低的趋势，CK在30d时达到最高，而100µM ABA处理后提前在20d达到最高，且种子的萌发也随之推迟。构建pTRV2-TgWRKY24沉默载体真空浸透侵染种子后，TgWRKY24基因表达量较未侵染的CK明显下降，种子的萌发提前。以100µM ABA处理沉默种子后的TgWRKY24表达量上升最明显，进一步说明了TgWRKY24基因的合成受ABA诱导。利用二代转录组与全长转录组联合分析筛选了ABA信号转导通路中的关键差异基因24个，并通过qRT-PCR进一步得到与TgWRKY24基因在种子休眠解除过程中表达特性一致且差异显著的基因TgABI5，通过酵母双杂系统与双分子荧光互补（BiFC）实验进一步验证了TgWRKY24和 TgABI5存在蛋白互作。本研究初步阐明了TgWRKY24响应ABA调控天山郁金香种子休眠解除的分子机制，丰富了植物种子休眠调控机理，为进一步应用我国优异的郁金香种质资源奠定基础。