ABA信号调控TaLCYB表达影响小麦类胡萝卜素合成的分子机制研究

Carotenoid is one of the major pigments and determines the nutritional value of wheat. However, the precise carotenoid metabolic engineering in crop plants has been hindered by limited data about the endogenous regulation of carotenogenic genes. Previously, we cloned and characterized the function of TaLCYB in wheat, which was key factor for the β-carotene accumulation in the transgenic wheat due to its high expression. There are several ABRE elements in the promoter of TaLCYB through sequence analysis. The expression of TaLCYB was down-regulated by the ABA treatment. Based on these results, we speculate that transcriptional modulation is crucial for ABA inducing the expression of TaLCYB gene. In this study, we will characterize the ABRE element in the promoter region of TaLCYB gene and their interactive transcription factors, study the roles and mechanisms involved in transcription factors modulating TaLCYB, and elucidate the molecular mechanism underlying ABA modulating the expression of TaLCYB gene to regulate the content of carotenoid. This work will be benefit for improving the understanding of the molecular mechanism of carotenoid regulation, and supply theoretical evidence for carotenoid metabolic engineering in wheat and other crops.

类胡萝卜素是小麦营养品质的重要组成部分，而类胡萝卜素合成代谢调控机制的匮乏限制了精准的类胡萝卜素代谢工程的应用进程。项目组前期研究在小麦中干扰TaLCYB基因显著降低了β-胡萝卜素含量，证实该基因是决定小麦籽粒中β-胡萝卜素含量的关键酶基因。进一步研究发现TaLCYB基因的启动子序列含有多个ABA应答元件，TaLCYB的表达显著受ABA信号抑制。因此，我们推测转录调控是ABA信号调控TaLCYB基因表达的重要方式，但具体分子机制仍不清楚。本研究将通过鉴定TaLCYB基因启动子上的ABA响应元件和与其互作的转录因子，研究ABA应答转录因子对TaLCYB基因的调控机理，以期阐明ABA调控TaLCYB基因表达进而影响β-胡萝卜素的含量和总类胡萝卜素积累的分子机制。研究结果将加深对类胡萝卜素合成调控机制的理解，为小麦及其它植物中的类胡萝卜素遗传代谢工程提供理论依据。

类胡萝卜素不仅是小麦营养品质的重要组成部分，也是参与植物发育与光响应的重要色素。尽管类胡萝卜素代谢酶已基本清楚，但是其上游调控因子的研究很少。为此，本研究以小麦中类胡萝卜素重要调控酶基因TaLCYB及其受ABA调控为切入点，通过酵母单杂筛库，得到了16个能与TaLCYB基因启动子互作的转录子基因；进一步结合公共数据库中小麦组织发育的RNA-seq数据构建的共表达网络进行分析，鉴定得到5个与TaLCYB-6A/6B/6D均呈现表达负相关的转录因子基因，ABA处理也证明ABA能显著诱导这5个转录因子基因的表达下调，与TaLCYB受ABA抑制的表达模式类似。综合运用生物信息分析和生化分子生物学实验手段研究了TaABF2和TaMYB106与TaLCYB启动子的互作，初步解析了它们调控TaLCYB表达的机制。亚细胞定位和转录激活活性结果显示TaABF2和TaMYB106都具有转录激活活性并定位于细胞核。创制了TaABF2、TaMYB106过表达和RNAi的转基因小麦材料，为小麦中类胡萝卜素代谢的转录调控和分子育种改良提供了新遗传材料。此外，我们系统鉴定了小麦类胡萝卜素代谢基因，通过转录组-代谢组联合分析解析了参与种子类胡萝卜素含量快速下降的多个关键代谢酶，包括DXR、GGPPS、CCD1和HYD1，为进一步改良种子类胡萝卜素相关性状提供了新基因资源。