HDA9, a member of the deacetylase family, plays a vital role in regulating plant flowering time through flowering integrator SOC1 and AGL24. However, it remains elusive how HDA9 interacts with SOC1 and AGL24 in flowering time control. Here, HDA9 was cloned in Brassica juncea and then its three active sites were separately replaced with Ala via overlap extension PCR. Thus, mutants of HDA9(D172A), HDA9(H174A) and HDA9(D261A) were constructed and fused into the pGADT7 vector. The yeast one-hybrid assays indicated that HDA9 mutants remained the interactions with the promoters of SOC1 and AGL24. Furthermore, the aforementioned results were confirmed in the dual luciferase assays. Interestingly, the DNA-protein interactions were weakened significantly due to the mutation in the three active sites of HDA9. It suggested that flowering signal integrator SOC1 and AGL24 were regulated by the key amino acid residues of 172th, 174th and 261th in HDA9. Our results provide valuable information for the in-depth study of the biological function and molecular regulation of HDA9 in Brassica juncea flowering time control.
芥菜 HDA9 是去乙酰化酶家族成员，能通过开花信号整合子 (SOC1、AGL24) 调控开花时间，但其深入的分子调控机制仍不清楚。利用重叠延伸PCR 将芥菜HDA9 的3 个关键活性位点 (Asp172、His174 和Asp261) 分别突变为Ala，构建氨基酸位点突变体HDA9(D172A)、HDA9(H174A) 和HDA9(D261A)。进一步将突变体融合到pGADT7 载体，酵母单杂交表明，HDA9 突变后仍能与开花整合子SOC1、AGL24 的启动子结合。双荧光素酶系统深入检测发现，尽管HDA9(D172A)、HDA9(H174A) 和HDA9(D261A) 与SOC1、AGL24 启动子的结合仍存在，但作用强度均显著减弱。由此暗示，芥菜HDA9 的第172、174 和261 这3 个关键活性位点可在一定程度上调节它与开花整合子的相互作用。这为HDA9 开花分子调控及功能解析等深入研究奠定了基础。.
Keywords: Brassica juncea; HDA9; flowering signal integrator; interaction.