牡丹PsBGs基因响应赤霉素参与休眠解除中运输通道重新打通的机制

To break dormancy is the keystone to the anti-season production of tree peony, and together with sufficient nutrient supplement is the guarantee to flowering quality. However, callose deposits both at plasmodesmata (PD) and sieve pore when plants go into dormancy. As a result, all symplasmic pathways are shut down, which will finally reduce flowering quality if they can’t be thoroughly reopened when dormancy release. It was proved that chilling and gibberellins could induce the synthesis of β-l,3-glucanases to degrade callose and recover the symplasmic paths. Previously, we had screened several β-l,3-glucanases genes (PsBGs) induced by chilling and GA, combining transcriptome and expression analysis. In this study, PsBGs genes involved in recruiting transport conduits will be identified by functional, in situ hybridization and localization analysis. Accordingly, distributions of callose at PD and sieve pore, activities of β-l,3-glucanase enzyme and alterations of transport paths during dormancy release are also evaluated to analysis their co-relationship with PsBGs expression. The promoters of PsBGs are cloned, and factors influencing their expression are explored using engineered Arabidopsis. GA response elements in the PsBGs will be identified by deletion analysis and mutation analysis in transgenic Arabidopsis, according to GUS activities. The transcriptional factors involved in regulation PsBGs by GA are then screened by yeast-one-hybridization and identified by EMSA and ChIP. The expression patterns of the identified transcriptional factors are studied, and the regulation mechanism of PsBGs by GA is finally elucidated. These results are of great value to understand the mechanism of dormancy release, the regulation of substance transport during dormancy release, and as an important basement to innovate the forcing culture technology for tree peony.

休眠的彻底解除、充足的营养供应是牡丹反季节生产中开花质量的保障。然而，内休眠伴随着胼胝质在筛孔和胞间连丝的沉积，从而堵塞了物质运输的通道，限制了营养的供应。低温和赤霉素（GA）可诱导β-l,3-葡聚糖酶的合成，降解胼胝质从而打通物质运输通道。课题组前期通过转录组测序结合表达模式分析克隆了受GA和低温诱导的β-l,3-葡聚糖酶基因（PsBGs），本项目拟通过功能分析、原位杂交和蛋白定位分析等鉴定参与运输通道重新打通的PsBGs基因；测定牡丹花芽休眠解除中胼胝质分布、β-l,3-葡聚糖酶活力和运输通路状态变化，分析其与相关PsBGs基因表达的相关性；进一步，通过缺失分析鉴定PsBGs启动子中的GA响应元件，并筛选、鉴定相关转录因子，解析GA诱导PsBGs基因表达的调控机制。研究结果对深入理解休眠解除中物质运输的调控，解析休眠解除机理具有重要价值，并为牡丹反季节催花生产技术创新提供理论依据。

牡丹（Paeonia suffruticosa Andr.）反季节催花是其产业的主要内容之一。休眠的彻底解除、充足的营养供应是牡丹反季节生产中开花质量的保障。然而，内休眠伴随着胼胝质在筛孔和胞间连丝的沉积，从而堵塞了物质运输的通道。本项目以牡丹品种‘鲁荷红’为材料，分析了休眠进程中花芽形态和运输通道的变化，鉴定了参与休眠调控相关的葡聚糖酶基因，分析了调控机制。低温累积进程中，牡丹花芽继续发育，直径不断增加，萼片、花瓣、雄蕊和心皮不断伸长，其表皮角质层和气孔数量增多，休眠的解除伴随着花瓣侧脉维管系统的建立。伴随着低温积累，花芽内葡聚糖酶活力逐渐升高，葡聚糖含量逐渐下降；饲喂钙黄绿素后，最初只能在花芽基部检测到荧光，21 d时顶端才出现荧光信号；苯胺蓝染色发现，枝条韧皮部也是逐渐打通的。赤霉素处理可诱导葡聚糖酶活力增加，葡聚糖含量下降，打通物质运输通道。根据转录组数据结合RACE扩增，获得9个β-1,3-葡聚糖酶基因，大都含有GH17和X8结构域。低温累积促进了PsBG1、PsBG3、PsBG6、PsBG7、PsBG8和PsBG9的上调表达，并得到原位杂交证实；GAs促进了PsBG1、PsBG3、PsBG8 和PsBG9的表达。构建了PsBGs的原核表达载体，诱导表达了重组蛋白，以昆布多糖酶为底物，HPLC发现重组的PsBGs具有葡聚糖酶活性。超表达PsBGs侧枝较多，根长相对较长。亚细胞定位证明，PsBG6、PsBG9基因定位于细胞之间的胞间连丝处。利用TAIL-PCR扩增了PsBG9启动子，获得了1500 bp的启动子序列，含有2个可能的Pyrimidine box元件，启动子分析证明其参与了赤霉素响应，酵母单杂获得了与其结合的ZFP转录因子，EMSA和ChIP实验证明二者的互作关系，qPCR分析表明ZFP可受低温和GA3的诱导。本项目结果对于深入理解内休眠中运输通道的调控奠定了基础。