14-3-3蛋程中的互作靶蛋白鉴定及其对外源激素的响应

【目的】籽粒灌浆对水稻产量及品质的形成至关重要。14-3-3蛋白是一种信号转导调节因子,在植物生长发育中发挥着重要调控作用。本研究通过分析14-3-3蛋白家族在籽粒灌浆过程中的基因表达模式及其互作靶蛋白,从而揭示其在籽粒灌浆过程中的功能。【方法】利用实时荧光定量PCR（qRT-PCR）技术分析水稻14-3-3基因家族在籽粒灌浆过程中的表达变化模式,并从中选取GF14b及GF14e进行后续的蛋白功能分析。利用KEGG数据库对GF14b及GF14e蛋白功能motif位点进行分析;构建GST-GF14b及GST-GF14e表达载体,利用亲和层析技术分别钓取籽粒中与GF14b及GF14e互作的靶蛋白,并借助LC-MS/MS对靶蛋白进行鉴定。采用GST pull-down方法验证靶蛋白与GF14b及GF14e间的蛋白互作关系。利用Kinasephos在线程序对靶蛋白的Ser和Thr磷酸化位点进行预测;采用MapMan 3.6.0软件对靶蛋白的功能及参与的代谢过程进行分析。在籽粒灌浆期（花后15 d）,分别喷施25×10^-6mol·L^-1 ABA,10×10^-6mol·L^-1 IAA,100×10^-6mol·L^-1 GA,50×10^-6mol·L^-1 ZR和 2×10^-4mol·L^-1 BR,研究外源激素处理对籽粒灌浆过程中GF14b,GF14e及其互作靶基因表达的影响。【结果】14-3-3家族基因中,除GF14h外,其余7个家族成员在水稻籽粒中均有表达,其中GF14b及GF14e在籽粒灌浆过程中的表达水平较高且变化幅度较大。通过蛋白序列分析发现,GF14b与GF14e间具有3个相同,2个差异的motif功能位点。通过亲和层析试验,在籽粒中共鉴定到59个与GF14b和72个与GF14e互作的靶蛋白,其中有43个靶蛋白与2个成员均有互作,分别有16个和29个靶蛋白与GF14b和GF14e特异结合。随机选取2个靶蛋白进行体外蛋白互作验证,结果表明靶蛋白SUS3与GF14b和GF14e均存在互作关系,而靶蛋白PSA仅与GF14e有相互作用关系,验证了亲和层析结果的准确性。蛋白功能的分析表明,GF14b和GF14e通过与靶蛋白的结合,共同参与了籽粒灌浆过程中蔗糖转化、淀粉合成、糖酵解、TCA循环等碳代谢途径。同时,GF14b及GF14e还具有特异的调控功能,其中GF14b与核酸代谢及物质转运密切相关,而GF14e与C1代谢中的关键蛋白存在互作。此外,大部分靶蛋白均鉴定到具有潜在的Ser和Thr磷酸化位点。外源激素处理下,籽粒中GF14b和GF14e上调表达,而与淀粉合成代谢相关的靶基因（SUS2、 AGPS、AGPL、PPDK2、SBE）大部分呈下调表达的趋势。【结论】14-3-3基因家族成员GF14b和GF14e在水稻籽粒灌浆过程中的表达变化幅度较大,且会响应激素浓度的改变,并通过蛋白互作的形式负调控淀粉合成代谢相关基因的表达,从而对水稻籽粒淀粉的合成起到重要的调控作用。

Identification of 14-3-3 Client Proteins in Rice Grains and Their Response to Exogenous Hormones During the Grain Filling Stage

【Objective】Grain yield and quality of rice mainly depend on grain filling. The family of 14-3-3 proteins is the important regulator of signaling in plant, and plays an important role in plant growth and development. This study was performed to assess the gene expression pattern and to identify client proteins, which would result in important information toward understanding the functional mechanism of 14-3-3 protein during rice grain filling. 【Method】 The expression pattern of each 14-3-3 genes at different grain filling stages were examined by the Real-time RT-PCR (qRT-PCR), and the family members GF14b and GF14e were used for subsequent functional analysis. The function motif of GF14b and GF14e protein were analyzed by using the KEGG database. The amplified open reading frames of GF14b and GF14e were cloned into the PGEX-6P-1 expression vector for production of the N-terminal-tagged fusion proteins GST-GF14b and GST-GF14e. The client proteins of GF14b and GF14e were captured with the affinity chromatography approach and identified by the LC-MS/MS. The protein interaction between client proteins and GF14b and GF14e were validated by the GST Pull-down in vitro. Phosphorylation sites and functions of all client proteins were analyzed using the KinasePhos online website and MapMan software (Version 3.60), respectively. The exogenous hormones (25×10 ^-6mol·L^-1 ABA,10×10^-6mol·L^-1 IAA,100×10^-6mol·L^-1 GA,50×10^-6mol·L^-1 ZR and 2×10^-4mol·L^-1 BR ) were sprayed on rice grains at 15 days after flowering to investigate the effects of exogenous hormones treatment on the expression of GF14b, GF14e and their interacting genes. 【Result】 Except for the GF14h, the remaining seven 14-3-3 family genes were expressed in rice grains, and the expression of GF14b and GF14e was high and highly variable during grain filling. Two same protein motifs and three differential motifs between GF14b and GF14e were identified with the protein sequence analysis. In total, 59 GF14b- and 72 GF14e-client proteins in rice grains were captured by affinity chromatography on a glutathione-agarose affinity column. Two randomly selected proteins from these client proteins, namely SUS3 and PSA, were further validated by GST Pull-down in vitro, and the results showed that the SUS3 interacted with GF14b and GF14e, whereas PSA specially interacted with GF14e, which confirmed the accuracy of the affinity chromatography assay. Among these client proteins, fourth-three were commonly affinity-identified by the GF14b and GF14e and were predicted to be implicated in the sucrose conversion, starch synthesis, glycolysis and TCA cycle. Meanwhile, it was found GF14b specifically bounded to the nucleotide metabolism and transporters, whilst GF14e tended to interact with the proteins involving C1 metabolism. In addition, most of the identified client proteins possess the phosphorylation sites of Ser or Thr. The present results also showed that the expression of GF14b and GF14e were up-regulated and most of the starch synthesis-related interacting genes (SUS2, AGPS, AGPL, PPDK2, SBE) were down-regulated after exogenous hormones treatment. 【Conclusion】 The magnitude of change in gene expression was greater in GF14b and GF14e than the other 14-3-3 gene family members during grain filling. The GF14b and GF14e could respond the change of hormones concentration and act as a regulator to negatively regulate the expression of starch synthesis related genes through protein-protein interaction, in turn, play an important role in starch synthesis during the grain filling stage.