拟南芥G蛋白α亚基GPA1互作蛋白铜离子结合蛋白AtBCB的鉴定及功能分析

拟南芥G蛋白复合体(异源三聚体包括α、β、γ亚基)参与植物多个信号转导途径，G蛋白复合体通过膜上的G蛋白偶联受体(GPCR)接受胞外信号后通过3个亚基将信号传递给下游效应器。目前，有关植物G蛋白复合体的效应器及其信号传递途径的报道较少，寻找新的G蛋白的效应器有助于阐明G蛋白复合体相关的信号传导途径。本研究以拟南芥G蛋白α亚基GPA1为诱饵蛋白，利用泛素分离系统筛选拟南芥cDNA文库，获得一个与GPA1互作的铜离子结合蛋白AtBCB。荧光双分子杂交(BiFC)试验证明，GPA1与AtBCB的互作发生在细胞膜上。基因表达特性分析结果显示，GPA1和AtBCB受金属铝胁迫的诱导表达。进一步以野生型拟南芥(WT)、GPA1拟南芥突变体gpa1-4和AtBCB拟南芥突变体bcb为材料，研究该基因对植物耐金属铝胁迫的功能，结果显示，在无胁迫情况下，2个突变体和WT根部的丙二醛含量无显著差异；在100 µmol L^–1 Al³⁺处理下，gpa1-4突变体根部丙二醛含量显著(P<0.05)低于WT低；bcb根部丙二醛含量极显著(P<0.01)高于WT。对3个铝胁迫响应基因(苹果酸转运体基因AtALMT1、半类型ABC转运蛋白基因ALS1和ABC转运蛋白基因ALS3)的表达进行Real-time PCR分析，比较它们在突变体和野生型之间的表达差异，发现在有铝和无铝处理情况下，ALS1和ALS3的表达水平在突变体和WT间均无显著差异；在铝处理下，gpa1-4中AtALMT1的表达量极显著高于WT；在bcb中的表达量显著低于WT。以上结果表明，植物通过细胞膜上的G蛋白α亚基GPA1和铜离子结合蛋白AtBCB的相互作用调控下游基因AtALMT1的表达，参与植物对铝胁迫的响应，其中GPA1对铝胁迫耐受起负向作用，AtBCB对铝胁迫耐受起正向作用。

Characteristic and Function Analysis of a Copper Ion Binding Protein,AtBCB Interacting with G Protein α Subunit GPA1 in Arabidopsis thaliana

Heterotrimeric G protein, including three subunits of α, β, and γ, is involved in a lot of signaling pathways in plants. It receives extracellular signals via G-protein coupled receptor (GPCR) and transmits them to the downstream effectors by the three subunits. Till now, the downstream effectors and signaling pathway related to G-protein complexes have been rarely reported. Furthermore, identifying more novel G protein effectors would be helpful to elucidate signaling pathway associated with the G protein complex. In order to find some novel effectors, G protein α subunit (GPA1) was used as bait to screen interaction protein in Arabidopsis by the split-ubiquitin screening system in this study. One of the GPA1-interacting proteins was identified as copper ion binding protein,AtBCB. The interaction betweenGPA1 and AtBCB was verified on cell membrane by BiFC (bimolecular fluorescence complementation). The expressions of GPA1 and AtBCB were confirmed to be induced by aluminium stress. To study the function of the two genes, we treated Arabidopsis mutant gpa1-4 and bcb, in which GPA1 and AtBCB were knocked out, with 100 µmol L^–1 Al³⁺, respectively, and then measured MDA (malonaldehyde) content in roots. The results showed that MDA content in both mutant and WT (wild type) under normal condition was no significant difference, but when exposured to 100 µmol L^–1 Al³⁺ the content in gpa1-4 was lower than that in WT (P<0.05), and it in bcb was higher than that in WT (P<0.01). Furthermore, the expression patterns of three responsive genes ofALMT1, ALS1 and ALS3 to aluminum toxicity were analyzed by Real-time PCR. The results showed that no matter the condition with or without aluminum stress, the expression of ALS1 and ALS3 appeared no significant difference in the mutants and WT. However, in the treatment with less than 100 µmol L^–1 Al the expression level of ALMT1 ingpa1-4 was higher than that in WT, and the expression level of ALMT1 inbcbwas lower than that in WT. In short, GPA1 and AtBCB directly interact in the cell membrane, and regulate the expression of the downstream gene of ALMT1. In the tolerance process to aluminum stress in plants, GPA1 plays negative role but AtBCB has positive effect.