关键词: 水稻, 籽粒灌浆, 14-3-3蛋白, 蛋白互作, 激素

Abstract:

【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.

Key words: rice, grain filling, 14-3-3 protein, protein-protein interactions, hormone