芥菜开花整合子SOC1与AGL24蛋白K结构域的互作位点鉴定

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

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AGL24^(R137L)和AGL24^(E169L)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(AGL24^(Q107L)),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:SOC1^(V77K)、SOC1^(P81K)、SOC1^(K108V)、SOC1^(R109L)和SOC1^(C137K)突变体均能与AGL24相互作用;但SOC1^(V77K)、SOC1^(P81K)、SOC1^(K108V)和SOC1^(R109L)突变体与AGL24的作用强度均显著低于SOC1/AGL24,而SOC1^(C137K)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。

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 SOC1^(V77K),SOC1^(P81K),SOC1^(K108V),SOC1^(R109L) and SOC1^(C137K))and three single-site mutants of AGL24(respectively named AGL24^(Q107L),AGL24^(R137L) and AGL24^(E169L))to unravel the molecular mechanism of the protein interactions of SOC1 and AGL24. Yeast two-hybrid experiments and β-galactosidase activity assays showed that mutant of AGL24^(Q107L) could no more interact with SOC1. However,AGL24^(R137L) and AGL24^(E169L) 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 137 th or 169 th but 107 th in the K-domain of AGL24. Further research showed that mutants of SOC1^(V77K),SOC1^(P81K),SOC1^(K108 V),SOC1^(R109L) and SOC1^(C137K) remained interacting with AGL24 protein. Mutants of SOC1^(V77K)、SOC1^(P81K)、SOC1^(K108V) and SOC1^(R109L) fused with AGL24 had significantly low interaction strength compared with SOC1/AGL24. On the contrary,the interaction strength of SOC1^(C137K) 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 77 th,81st,108 th and 109 th amino acids,as well as positive regulation site of 137 th 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.