异源表达N-端融合eYFP标签的GAAPs增强拟南芥对ER胁迫敏感性

Ectopic expression of GAAPs tagged with eYFP at the N-terminal in Arabidopsis enhanced plant sensitivity to endoplasmic reticulum stress

高尔基体抗凋亡蛋白GAAPs(Golgi anti-apoptotic proteins)在真核生物中高度保守,是动物细胞中抑制胁迫诱导凋亡的BAX inhibitor-1类因子。反向遗传学研究表明,拟南芥GAAPs也能够抑制ER胁迫诱导的细胞死亡。为进一步在蛋白质水平研究GAAPs的功能和作用机制,通常使用基因融合标签的表达转化植株,但要考虑所使用的标签是否影响蛋白的功能。为此,我们分别构建了GAAP1和GAAP4的N末端融合e YFP、无e YFP标签的拟南芥过量表达转化子。通过检测e YFP-GAAP1/GAAP4以及gaap1突变体对ER胁迫敏感性,发现e YFPGAAP1/GAAP4过量表达与gaap1突变体类似,ER胁迫下的存活率降低,细胞死亡增强。定量PCR分析表明e YFPGAAP1过量表达抑制急性或长期ER胁迫下未折叠蛋白响应(unfolded protein response,UPR)通路相关基因的表达。这些结果表明GAAPs的N-端融合e YFP标签影响其在ER胁迫抗性以及抗细胞死亡中的功能。

Golgi anti-apoptotic proteins（GAAPs） are highly conserved throughout eukaryotes and GAAP resembles BAX inhibitor-1 with inhibiting apoptosis triggered by various stresses in mammalian cells.Arabidopsis GAAPs have also been showed inhibited cell death induced by endoplasmic reticulum（ER） stress by reverse genetic assay.Except for the mutants,the transgenic plants expressing the gene fused with different tags are usually generated to study the function and its mechanism.But it should be caution whether the tag affect the protein function firstly.In the present study,we generated Arabidopsis plants overexpressing GAAP1 or GAAP4 tagged with e YFP at their N-terminal respectively.The sensitivity of e YFP-GAAP1/GAAP4 and gaap1 mutants to the ER-stress was tested.And the results showed that overexpressing e YFP-GAAP1/GAAP4 reduced the plant survival and enhanced cell death under ER stress,which was similar as gaap1 mutants.The quantitative real time PCR analysis showed that overexpressing e YFP-GAAP1 inhibited the activation of unfolded protein response（UPR） signaling pathway genes upon acute or prolonged ER stress.These data suggested that the e YFP tag at the N-terminal of GAAPs affected its function in the resistance of ER stress and anti-programmed cell death.