摘要
Protein kinase C (PKC) has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina (DsPKC) using rapid amplification of cDNA ends (RACE) and RT-PCR methods. And we submitted the mRNA sequence of DsPKC gene to NCBI (Genbank No. JN625213). In the present paper, the DsPKC gene open reading frame obtained by PCR was cloned into pGS-21a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The DsPKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.2 mmol L 1 at 37℃. Under salt stress, the fu- sion protein Green Fluorescent Protein (GFP)-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pat- tern of DsPKC gene was analyzed using real-time quantitative PCR, and indicated that DsPKC gene was up-regulated by 3.0 mol L 1 NaCl at 12 h, which was significantly higher than in control values (P < 0.05). These results suggest that the DsPKC gene plays an important role in response to salt stress in D. salina.
Protein kinase C（PKC） has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. We previously isolated the full-length PKC gene from Dunaliella salina（Ds PKC） using rapid amplification of cDNA ends（RACE） and RT-PCR methods. And we submitted the mRNA sequence of Ds PKC gene to NCBI（Genbank No. JN625213）. In the present paper, the Ds PKC gene open reading frame obtained by PCR was cloned into pGS-21 a vector and transformed into Escherichia coli to generate the fusion protein. Bioinformatics analysis revealed that DsPKC gene was a member of serine/threonine kinase with two conserved domains and highly conserved motifs. The Ds PKC was highly expressed upon induction with isopropyl-β-d-thiogalactoside（IPTG） at a final concentration of 0.2 mmol L-1 at 37℃. Under salt stress, the fusion protein Green Fluorescent Protein（GFP）-DsPKC was transferred from the cytoplasm to the cell membrane. The expression pattern of Ds PKC gene was analyzed using real-time quantitative PCR, and indicated that Ds PKC gene was up-regulated by 3.0 mol L-1 NaCl at 12 h, which was significantly higher than in control values（P < 0.05）. These results suggest that the Ds PKC gene plays an important role in response to salt stress in D. salina.
基金
the functional analysis of PKC signaling pathway involved in response to salt stress of Dunaliella salina
the National Natural Science Foundation of China (No. 31472260)
