CsbHLH113转录因子调控柑橘果实柠檬酸代谢机制解析

The bHLH transcription factor family has diverse and crucial functions in higher plants. A transcription factor CsbHLH113 showing a role in regulating citrate metabolism was found in our previous research. However, the specific mechanism of CsbHLH113 in citrate regulation is unknown. . In this project, we will first confirm the gene function in citrate regulation through transient expression of CsbHLH113 in tobacco and by transforming CsbHLH113 into embryogenic citrus callus. The effect of CsbHLH113 on other metabolites in transgenic materials will be analyzed as well. Afterwards, interactions between CsbHLH113 and genes or transcription factors that are involved with citrate metabolism in citrus will be detected using a yeast-one- or yeast-two-hybrid system. Finally, other genes that are involved in citrate metabolism and function through CsbHLH113 regulation will be revealed using ChIP-Seq. . With this project, a regulation network on citrus citrate metabolism mediated by CsbHLH113 can be constructed. Results of this research contribute to understanding of citrate metabolism in citrus and lay the foundation for improving fruit quality.

bHLH基因家族在植物中是一类功能广泛、作用重要的转录因子。本项目前期在柠檬酸代谢研究中筛选到一个调控柑橘果实柠檬酸代谢的转录因子CsbHLH113，其调控柠檬酸代谢的具体机制尚不清楚。本项目首先将CsbHLH113转录因子转化烟草和柑橘胚性愈伤组织，验证其调控柠檬酸代谢的功能及其对其他代谢物的影响；进而通过酵母单杂交和双杂交系统对CsbHLH113转录因子与已知柠檬酸代谢关键功能基因和转录因子的互作关系进行定向检测；然后通过ChIP-Seq的方法在基因组水平筛选CsbHLH113转录因子调控的其他参与柠檬酸代谢的基因，最终揭示CsbHLH113转录因子介导的柑橘果实柠檬酸代谢调控网络。本项目的开展，对完善柑橘柠檬酸代谢机制具有重要意义，也为进一步调节果实品质形成奠定基础。

有机酸是决定果实风味的重要组成之一，柠檬酸是柑橘果实中主要的有机酸。本项目基于前期筛选到的一个参与柑橘果实柠檬酸代谢的转录因子CsbHLH113，从分子生物学和遗传学水平鉴定CsbHLH113在果实柠檬酸积累中的功能，挖掘CsbHLH113介导的柑橘果实柠檬酸代谢调控网络。研究结果显示，CsbHLH113在高酸品种‘大红甜橙’(DH)中较低酸品种‘冰糖橙’(BT)显著上调表达，亚细胞定位显示其定位于细胞核。以冰糖橙、烟草和番茄为材料鉴定基因功能结果表明，CsbHLH113瞬时超表达转化冰糖橙汁胞及烟草后，各组织柠檬酸含量分别升高103%和56%；过表达CsbHLH113番茄植株较WT提早开花、坐果和转色，超表达番茄株系柠檬酸含量较WT分别显著升高14-60%。通过酵母单杂交及双荧光素酶系统鉴定发现柠檬酸积累关键基因CsPH8为CsbHLH113调控的靶基因，柑橘叶片过表达CsbHLH113基础上沉默CsPH8后，柠檬酸含量仍显著降低23%。综上，CsbHLH113转录因子通过调控CsPH8的表达从而介导柑橘果实柠檬酸积累。依托本项目共发表论文10篇，培养博士研究生2人，硕士研究生9人。