芥菜开花整合子SOC1与AGL24蛋白K结构域的互作位点鉴定

芥菜开花整合子SOC1与AGL24相互作用能够调节开花时间。为了深入研究SOC1与AGL24蛋白互作的分子机理，利用ISIS系统在线预测了SOC1/AGL24互作位点，并分别在MIKC型蛋白SOC1和AGL24的K域构建了5个SOC1突变体和3个AGL24突变体。酵母双杂交和β–半乳糖苷酶活性检测表明：突变体AGL24R137L和AGL24E169L能够与SOC1蛋白互作，但作用强度与SOC1/AGL24差异不显著；然而AGL24蛋白第107位的谷氨酰胺突变为亮氨酸后（AGL24Q107L），则与SOC1的作用消失。说明SOC1/AGL24的作用强度能够被AGL24蛋白K域第107位调节，但可能不受第137和169位调控。进一步研究发现：SOC1V77K、SOC1P81K、SOC1K108V、SOC1R109L和SOC1C137K突变体均能与AGL24相互作用；但SOC1V77K、SOC1P81K、SOC1K108V和SOC1R109L突变体与AGL24的作用强度均显著低于SOC1/AGL24，而SOC1C137K突变体与AGL24的作用显著高于SOC1/AGL24。说明SOC1/AGL24的作用强度能够被SOC1蛋白K域第77、81、108、109位负调控或者第137位正调控。这为利用氨基酸位点调节SOC1/AGL24的深入研究及其开花时间分子调控奠定了基础。

Identification of Interaction Sites in K-domains of Flowering Signal Integrator SOC1 and AGL24 in Brassica juncea

The flowering signal integrator SOC1 can interact with AGL24 in Brassica juncea，which plays an important role in regulating flowering time. Based on the binding sites in the K-domains of MIKC-type proteins predicted by ISIS system，we constructed five single-site mutants of SOC1（respectively named SOC1V77K，SOC1P81K，SOC1K108V，SOC1R109L and SOC1C137K）and three single-site mutants of AGL24（respectively named AGL24Q107L，AGL24R137L and AGL24E169L）to unravel themolecular mechanism of the protein interactions of SOC1 and AGL24. Yeast two-hybrid experiments and β-galactosidase activity assays showed that mutant of AGL24Q107L could no more interact with SOC1. However，AGL24R137L and AGL24E169L mutants as well as AGL24 retained similarly strong interacting with SOC1 protein. So it was suggested that the interactions of SOC1/AGL24 were probably not regulated by the amino acid sites of 137th or 169th but 107th in the K-domain of AGL24. Further research showed that mutants of SOC1V77K，SOC1P81K，SOC1K108V，SOC1R109L and SOC1C137K remained interacting with AGL24 protein. Mutants of SOC1V77K、SOC1P81K、SOC1K108V and SOC1R109L fused with AGL24 had significantly low interaction strength compared with SOC1/AGL24. On the contrary，the interaction strength of SOC1C137K and AGL24 was greatly higher than that of SOC1/AGL24. It was indicated that the interaction strength of SOC1/AGL24 could be controlled by negative regulation sites of 77th，81st，108th and 109th amino acids，as well as positive regulation site of 137th amino acid in the K-domain of SOC1 protein. The study provided valuable information for further studies on direct regulation of SOC1/AGL24 and molecular mechanisms of flowering time in Brassica juncea.