Differentially expressed protein and gene of a freshwater alga, Spirogyra varians, which is grown under two different temperatures (4℃ and 20℃) was analyzed. About 21 proteins were up-regulated and 7 proteins newly appeared at 4℃ and 9 proteins were significantly down-regulated at this condition. Co...
Differentially expressed protein and gene of a freshwater alga, Spirogyra varians, which is grown under two different temperatures (4℃ and 20℃) was analyzed. About 21 proteins were up-regulated and 7 proteins newly appeared at 4℃ and 9 proteins were significantly down-regulated at this condition. Cold regulated proteins are related to photosynthetic machinery (ELIP-like protein), protein synthetic pathway (26S proteosome regulatory subunit S5A, Eukaryotic initiation factor 4A), Energy transfer (NAD-FAD binding protein, ATP synthase) and production of antioxidant materials (vitamin synthetic pathway, Thiazole biosynthetic enzyme). Two genes which are most reproducible (ELIP-like protein, Thiamine biosynthetic enzyme) were isolated on 2DE and were named as SVCRs (Spirogyra varians cold regulated protein). A 20 KDa protein, SVCR1, (pI 4.5) which was most strongly up-regulated at 4℃ (about 500-fold higher than that at 20℃) was isolated. A partial amino acid sequence of this protein was obtained using Ettan-MALDI TOF mass spectrometry. A cDNA encoding SVCR1 was cloned using degenerated primers designed based on an internal amino acid sequence of the protein and λZAP cDNA library. This cDNA consisted of 745bp containing a 549bp open reading frame (ORF) and 47bp/149bp of 5'/3'-untranslated region respectively. The deduced amino acid had a high sequence similarity with early light-inducible protein (ELIP) which is known as nuclear-encoded chloroplast protein induced by light stress. This gene was targeted to chloroplast by signal peptide and is predicted to possess three transmembrane regions. The northern blot results showed that the accumulation of SVCR1 transcripts could be induced by cold treatment (4℃) even in the dark. Moreover, the SVCR1 was rapidly accumulated at both high light (1200 μmol photon m^(-2)s^(-1)) and UV-B irradiates condition (within 3hrs). Red light and below 1000 μmol photon m-2s-1 light did not affect the transcription of SVCR1. The SVCR1 transcription level was reduced by increase of Carbon dioxide concentration at 4℃. During incubation at 4℃ for several days, total pigments contents (Chl a, b, zeaxanthin, and beta-carotene) were reduced. A 35kDa protein, SVCR2, was isolated and sequenced using MALDI MS/MS TOF spectroscopy. It has a similarity with THI4 gene which is involved in vitamin biosynthetic pathway in higher plants. This gene was transcribed under low-temperature combination with light. During transcription of SVCR2 gene, antioxidant levels in methanolic extract increased more than 100 % at 4℃. An antioxidant enzyme activity (catalase and superoxide dismutase) was observed to be either unchanged or reduced during incubation at 4℃. Differentially produced antioxidants are isolated using High Perpormance Liquid Chromatography (HPLC). These compounds had a radical scavenging activity. Using DEG methods, four cold regulated genes were isolated and named as a gSVCRs (Spirogyra varians cold regulated gene group). The full length sequence was obtained using 5… and 3… RACE. They are shikimate ehydrogenase, transmembrane protein, RNA polymerase SIG 5 and unknown gene. The bi-functional 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQ/SDH, gSVCR1) gene of higher plants has two conserved domains of DHQase I and SDH (AroE) along with shikimate-binding site similar to other DHQ/SDH genes. Northern blot analysis showed that the gSVCR1 was up-regulated at the temperature below 4?XC. When combined with high-light stress (>50 μmol photon m-2s-1), the expression of gSVCR1 was highly intensified by low-temperature. The expression was not affected by light intensity up to 200 μmol photon m-2s-1 at the temperature higher than 10℃. The Shikimate dehydrogenase display using zymogram method indicated that there were three isozymes of DHQ/SDH in S. varians. The deduced amino acid sequence of gSVCR2 had a similarity with trans-membrane protein in Arabidopsis thaliana (Q9M2D2, 52.7%). Northern blot analysis demonstrated that the transcript level of SVCR2 increased about 10 fold under low temperature (4℃) compared with that cultured at warm (20℃) conditions. The expression of gSVCR2 was also affected by light conditions. When the plants were exposed to high light (1200 μmol photon m-2 s-1), the expression of gSVCR2 began within 2 hrs. This gene expression lasted for 4 hrs and decreased afterwards. Under the blue light (470 nm) condition, the expression of this gene was induced in same way as HL treatment, even under less than 100 μmol photon m^(-2) s^(-1). But red light (650 nm) and UV-A irradiation did not affect the expression of gSVCR2.
Differentially expressed protein and gene of a freshwater alga, Spirogyra varians, which is grown under two different temperatures (4℃ and 20℃) was analyzed. About 21 proteins were up-regulated and 7 proteins newly appeared at 4℃ and 9 proteins were significantly down-regulated at this condition. Cold regulated proteins are related to photosynthetic machinery (ELIP-like protein), protein synthetic pathway (26S proteosome regulatory subunit S5A, Eukaryotic initiation factor 4A), Energy transfer (NAD-FAD binding protein, ATP synthase) and production of antioxidant materials (vitamin synthetic pathway, Thiazole biosynthetic enzyme). Two genes which are most reproducible (ELIP-like protein, Thiamine biosynthetic enzyme) were isolated on 2DE and were named as SVCRs (Spirogyra varians cold regulated protein). A 20 KDa protein, SVCR1, (pI 4.5) which was most strongly up-regulated at 4℃ (about 500-fold higher than that at 20℃) was isolated. A partial amino acid sequence of this protein was obtained using Ettan-MALDI TOF mass spectrometry. A cDNA encoding SVCR1 was cloned using degenerated primers designed based on an internal amino acid sequence of the protein and λZAP cDNA library. This cDNA consisted of 745bp containing a 549bp open reading frame (ORF) and 47bp/149bp of 5'/3'-untranslated region respectively. The deduced amino acid had a high sequence similarity with early light-inducible protein (ELIP) which is known as nuclear-encoded chloroplast protein induced by light stress. This gene was targeted to chloroplast by signal peptide and is predicted to possess three transmembrane regions. The northern blot results showed that the accumulation of SVCR1 transcripts could be induced by cold treatment (4℃) even in the dark. Moreover, the SVCR1 was rapidly accumulated at both high light (1200 μmol photon m^(-2)s^(-1)) and UV-B irradiates condition (within 3hrs). Red light and below 1000 μmol photon m-2s-1 light did not affect the transcription of SVCR1. The SVCR1 transcription level was reduced by increase of Carbon dioxide concentration at 4℃. During incubation at 4℃ for several days, total pigments contents (Chl a, b, zeaxanthin, and beta-carotene) were reduced. A 35kDa protein, SVCR2, was isolated and sequenced using MALDI MS/MS TOF spectroscopy. It has a similarity with THI4 gene which is involved in vitamin biosynthetic pathway in higher plants. This gene was transcribed under low-temperature combination with light. During transcription of SVCR2 gene, antioxidant levels in methanolic extract increased more than 100 % at 4℃. An antioxidant enzyme activity (catalase and superoxide dismutase) was observed to be either unchanged or reduced during incubation at 4℃. Differentially produced antioxidants are isolated using High Perpormance Liquid Chromatography (HPLC). These compounds had a radical scavenging activity. Using DEG methods, four cold regulated genes were isolated and named as a gSVCRs (Spirogyra varians cold regulated gene group). The full length sequence was obtained using 5… and 3… RACE. They are shikimate ehydrogenase, transmembrane protein, RNA polymerase SIG 5 and unknown gene. The bi-functional 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQ/SDH, gSVCR1) gene of higher plants has two conserved domains of DHQase I and SDH (AroE) along with shikimate-binding site similar to other DHQ/SDH genes. Northern blot analysis showed that the gSVCR1 was up-regulated at the temperature below 4?XC. When combined with high-light stress (>50 μmol photon m-2s-1), the expression of gSVCR1 was highly intensified by low-temperature. The expression was not affected by light intensity up to 200 μmol photon m-2s-1 at the temperature higher than 10℃. The Shikimate dehydrogenase display using zymogram method indicated that there were three isozymes of DHQ/SDH in S. varians. The deduced amino acid sequence of gSVCR2 had a similarity with trans-membrane protein in Arabidopsis thaliana (Q9M2D2, 52.7%). Northern blot analysis demonstrated that the transcript level of SVCR2 increased about 10 fold under low temperature (4℃) compared with that cultured at warm (20℃) conditions. The expression of gSVCR2 was also affected by light conditions. When the plants were exposed to high light (1200 μmol photon m-2 s-1), the expression of gSVCR2 began within 2 hrs. This gene expression lasted for 4 hrs and decreased afterwards. Under the blue light (470 nm) condition, the expression of this gene was induced in same way as HL treatment, even under less than 100 μmol photon m^(-2) s^(-1). But red light (650 nm) and UV-A irradiation did not affect the expression of gSVCR2.
주제어
#Cold stress Cold regulated Differentially expressed gene Early light inducible protein Oxidative stress Photosynthesis Shikimate dehydrogenase Spirogyra varians SVCRs Thiamine biosynthetic enzyme
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