Cloning and Characterization of Gene Encoding the Regulator of G Protein Signaling Protein (BnRGS1) in Brassica Napus
|Course||Biochemistry and Molecular Biology|
|Keywords||Brassica napus BnRGS1protein G protein signaling plant hormone abiotic stress protein interaction|
G protein signaling exists widely in eukaryotes, and plays an important role in growth and development of plants and animals. Regulator of G protein signaling proteins (RGS) accelerate the rate of GTP hydrolysis by Ga proteins, thus acting as negative regulators of G-protein signaling. Studies in Arabidopsis and soybean have proven that RGS proteins are physiologically important in plants and contribute to the signaling pathways regulated by different stimuli. Besides rice and soybean, Brassica napus is another agriculturally relevant plant, the wildly planted oilseed rape in the world, which possesses an identified Gα, Gβ and Gγ subunits. The G protein of Brassica napus is involved in the response to plant hormones and abiotic stresses. There is no any RGS protein found in Brassica to date.In the present study, We have isolated a full-length cDNA from Brassica napus encoding proteins homologous to RGS, designated as BnRGS1. We have predicted and analyzed the structure of BnRGS1protein by bioinformatics methods. We also have studied the subcellular localization of the BnRGS1protein and verified the existence of the interaction between BnRGS1and BnGA1by the yeast two-hybrid system. The expression patterns of BnRGS1in various tissues and developmental stages and response to exogenous hormones treatment and abiotic stresses were analyzed by Q-PCR. The main results were as follows:We identified and characterized a Brassica napus RGS gene, BnRGS1. It contained an open reading frame of1380bp encoding a putative52.6-kDa polypeptide of459amino acids. BnRGS1protein has seven putative transmembrane domains in the N-terminal (amino acids1-294) and RGS box in the C-terminal (amino acids295-413). The deduced amino acid sequence of BnRGS1had a high degree of homology with those of RGS proteins from various biological sources, including Arabidopsis thaliana AtRGS1(88.10%), BrRGS1(84.10%), GmRGS1(63.30%) and GmRGS2(62.66%).BnRGS1was located on the membrane. The gene constructs for BnRGS1-YFP fusion proteins were introduced into epidermal cells of onion (Allium cepa) bulb by agrobacteria GV3101, and YFP fluorescence was observed in the plasma membrane. We performed plasmolysis of the cells; here, the YFP signal followed the plasma membrane when the protoplasts retracted from the cell wall. BnRGS1-YFP was also localized to membrane in tobacco leaves. Based on these results, we concluded that BnRGS1localized to the plasma membrane.BnRGS1interacts with BnGAl in Yeast. The protein-protein interaction between BnRGS1and BnGA1was examined in the mating-based split-ubiquitin system. There is a protein-protein interaction between BnRGS1and BnGAl.The expression levels of BnRGSl were quite different in different tissues and developmental stages. BnRGSl transcripts were predominantly accumulated in leaves, hypocotyls, roots and legumes, while less expressed in stems. The expression levels of BnRGSl at the two-leave stage, the seedling at the seventh day and the bolting stage were significantly higher than those at other developmental stages. The lowest expression level of BnRGSl transcripts was detected at four-leave stage. During rape seed germination, the highest expression of BnRGSl was detected in12h after germination, followed by a gradual decrease till36h.The expression levels of BnRGSl were different in response to plant hormones. When seedlings were treated with different ABA concentrations, the expression of BnRGS1increased with the increment of ABA concentration, and decreased with higher ABA concentrations. A similar relationship had also been observed when application with exogenous IAA. Low concentration of IAA significant increased the expression of BnRGSl, which was decreased with higher concentrations of IAA. Compared to these two hormones, the effects of GA3and BR on the expression of BnRGSl were unobvious and irregular under the concentrations tested.Besides plant hormone, the effects of some abiotic stresses were also investigated. When treated with low temperature at4℃, the expression of BnRGS1was increased gradually, which appeared inordinate with high temperate at40℃. The transcript level of BnRGSl was also induced by polyethylene glycol (PEG), whereas remained little changed by200mM NaCl. These results suggested that the BnRGSl gene may be involved in B. napus response to plant hormone signaling and abiotic stresses such as cold and drought.