MADS-box转录因子参与调控枇杷果实采后冷害木质化的机理研究

Loquat (Eriobotrya japonica) fruits are prone to exhibit chilling induced lignification during low temperature storage (0°C), with the increase of fruit firmness and accumulation of lignin content, which would subsequently influence fruit flavor and commercial value. The research on transcriptional regulation of chilling induced flesh lignification mainly focused on MYB and NAC family members, while other transcription factors are rarely concerned. According to previous results, MADS-box genes might be involved in flesh lignification, however, the mechanism remains unknown. Based on RNA-Seq, this project intends to characterize differentially expressed genes of MADS-box family in ‘Luoyangqing’ fruits (with different lignin contents). The transcriptional regulation effects of MADS-box family members will be analyzed by dual luciferase assay, yeast one hybrid system and EMSA. ChIP, SELEX and genome walking methods will be utilized to obtain the binding motif of key MADS-box members, in order to deeply mining the potential binding targets. Finally, transgenic technologies would be applied to verify biofunctions of key MADS-box members. The results of presented project will reveal the mechanism of how MADS-box genes get involved in chilling induced flesh lignification of loquat, providing novel evidence for improving postharvest technology of loquat industry.

枇杷果实低温冷藏易造成冷害木质化现象，表现为果实粗糙少汁，果肉中木质素含量上升，严重影响果实品质。目前，枇杷果实冷害木质化的转录调控研究以MYB和NAC转录因子为主，鲜有其他转录因子家族的报道。前期研究结果表明，枇杷MADS-box成员可能参与果实冷害木质化，但具体作用机制尚不明确。本项目拟利用RNA-seq技术分析木质化程度不同的‘洛阳青’枇杷果肉中MADS-box家族成员的表达模式，筛选冷害木质化相关MADS-box成员；结合双荧光素酶、酵母杂交、凝胶迁移技术分析其转录调控效应；采用染色质免疫共沉淀、指数富集的配体系统进化和染色质步移技术，深度挖掘MADS-box的潜在作用靶标，并通过转基因手段验证其功能，最终明确MADS-box成员参与冷害木质化的转录调控机制，为改进枇杷果实采后贮藏技术提供新的理论依据。

枇杷果实采后冷藏中极易发生木质化，表现为果实粗糙少汁，果肉中木质素含量上升，严重影响果实品质。理解冷害木质化发生的分子机制，有助于加快该产业问题的解决。目前，枇杷果实冷害木质化的机制研究关注于MYB和NAC等传统的次生细胞壁调控基因，而对于其他转录因子家族关注极少。申请人前期研究表明，MADS-box转录因子家族成员也参与果实冷害木质化，但具体作用机制尚不明确。本项目通过不同温度处理获得木质化程度不同的枇杷果实，通过RNA-seq技术分析了枇杷果肉中MADS-box家族成员的表达模式，筛选到冷害木质化相关MADS-box成员EjAGL65，双荧光素酶及酵母杂交结果表明，EjAGL65通过直接抑制结合EjMYB8启动子活性，以级联调控模式实现对木质素生物合成酶编码基因Ej4CL1转录过程的抑制，从而介入枇杷冷害木质化调控。在此基础上，进一步发现了另一个MADS-box成员EjAGL15，该基因显著诱导多个木质素生物合成途径基因的启动子活性，但仅在常温贮藏下高表达，表明MADS-box成员也参与了衰老引起的木质化调控。项目研究结果阐释了不同MADS-box亚家族成员独特的果肉木质化调控机制，证明了MADS-box家族成员深度参与枇杷采后木质化过程，拓展了对MADS-box基因传统生物学功能的认知，也深化了对枇杷采后木质化现象的机制理解，为解决枇杷采后贮藏产业问题提供了新的视角。