盐藻钙依赖蛋白激酶基因DsCDPK的表达分析
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摘要
以盐藻总RNA为模板,利用RT-PCR技术扩增了盐藻DsCDPK基因的cDNA序列,将其克隆到pMD~(TM)19-T simple载体上,经测序获得的克隆片段全长1650 bp,与已发表的盐藻DsCDPK(GenBank:JQ964113)的编码区序列同源性达100%。将DsCDPK基因的开放阅读框与质粒pET-32a(+)连接,构建原核表达载体pET-32a-DsCDPK。将该重组质粒转化到大肠杆菌BL21中,经IPTG诱导融合,蛋白在大肠杆菌BL21中得到成功表达。通过SDS-PAGE检测发现,融合蛋白为部分可溶性表达,将上清蛋白经过His柱纯化后获得了纯度较高的可溶性融合蛋白,Western杂交检测显示,融合蛋白能被抗His单克隆抗体特异性识别,初步证明该融合蛋白就是带有His标签的DsCDPK蛋白。采用实时荧光定量PCR方法分析了高盐胁迫下DsCDPK基因的表达模式。试验结果表明,盐藻DsCDPK基因为盐胁迫上调基因,在高盐(3.0 mol/L NaCl)胁迫下,DsCDPK基因表达量显著增加,盐胁迫1 h时表达量达到最高,为正常生长状况(1.0 mol/L NaCl)下的3倍,差异达到极显著水平(P<0.01)。该研究成果为进一步阐明盐藻钙依赖蛋白激酶基因的功能及作用机制奠定了基础。
In this study, cDNA corresponding to Ca~(2+)-dependent protein kinase(CDPK) gene(DsCDPK) was isolated from green alga Dunaliella salina using the total RNA of the green alga s as a template by reverse transcription PCR(RT-PCR), and the DsCDPK gene was cloned into pMDTM19-T simple vector. The DNA sequence of DsCDPK gene was shown to contain a 1650 bp open reading frame. Alignment analysis showed that the amino acid sequence of the gene was 100% identical to DsCDPK gene(GenBank No. JQ964113) summited in previous study. Then the DsCDPK gene was cloned into pET-32 a(+) expression vector, and introduced into Escherichia coli BL21(DE3) for expression. The fusion protein was induced with isopropyl β-D-1 thiogalactopyranoside(IPTG), and the soluble recombinant protein was expressed successfully and purified using a His-SefinoseTM Kit. The purified DsCDPK protein was identified further by SDS-PAGE and western blotting analysis. Real time quantitative RT-PCR demonstrated that DsCDPK was up-regulated genes with salt stress. The expression level of DsCDPK was induced by 3.0 mol/L NaCl, with the maximum in 1 h. The relative expression level of DsCDPK was 3-fold higher than that by 1.0 mol/L NaCl(P<0.01). These findings provide the bases for research of biological function and mechanism of DsCDPK gene.
In this study, cDNA corresponding to Ca~(2+)-dependent protein kinase(CDPK) gene(DsCDPK) was isolated from green alga Dunaliella salina using the total RNA of the green alga s as a template by reverse transcription PCR(RT-PCR), and the DsCDPK gene was cloned into pMDTM19-T simple vector. The DNA sequence of DsCDPK gene was shown to contain a 1650 bp open reading frame. Alignment analysis showed that the amino acid sequence of the gene was 100% identical to DsCDPK gene(GenBank No. JQ964113) summited in previous study. Then the DsCDPK gene was cloned into pET-32 a(+) expression vector, and introduced into Escherichia coli BL21(DE3) for expression. The fusion protein was induced with isopropyl β-D-1 thiogalactopyranoside(IPTG), and the soluble recombinant protein was expressed successfully and purified using a His-SefinoseTM Kit. The purified DsCDPK protein was identified further by SDS-PAGE and western blotting analysis. Real time quantitative RT-PCR demonstrated that DsCDPK was up-regulated genes with salt stress. The expression level of DsCDPK was induced by 3.0 mol/L NaCl, with the maximum in 1 h. The relative expression level of DsCDPK was 3-fold higher than that by 1.0 mol/L NaCl(P<0.01). These findings provide the bases for research of biological function and mechanism of DsCDPK gene.
引文
[1] 周丽,孟祥红,刘成圣,等.渗透胁迫对杜氏盐藻胞内甘油含量及相关酶活性影响[J].植物学通报,2006,23(2):145-151.
[2] Chen H,Lao Y M,Jiang J G.Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina[J].Science of The Total Environment,2011,409(7):1291-1297.
[3] Zhao L N,Gong W F,Chen X W,et al.Characterization of genes and enzymes in Dunaliella salina involved in glycerol metabolism in response to salt changes[J].Phycological Research,2013,61(1):37-45.
[4] He Q,Qiao D,Bai L,et al.Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina[J].Journal of Plant Physiology,2007,164(2):214-220.
[5] Jia Y,Xue L,Liu H,et al.Characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from the halotolerant alga Dunaliella salina and inhibition of its expression by RNAi[J].Current Microbiology,2009,58(5):426-431.
[6] Liska A J,Shevchenko A,Pick U,et al.Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics[J].Plant Physiology,2004,136(1):2806-2817.
[7] Katz A,Waridel P,Shevchenko A,et al.Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis[J].Molecular & Cellular Proteomics,2007,6(9):1459-1472.
[8] 陈硕,陈珈.植物中钙依赖蛋白激酶(CDPKs)的结构与功能[J].植物学通报,2001,18(2):143-148.
[9] Roberts D M,Harmon A C.Calcium-dependent proteins:target of intracellular calcium signals in higher plants[J].Annual Review of Plant Biology,1992(43):375-414.
[10] Johnson C H,Knight M R,Kondo T,et al.Circadian oscillations of cytosolic and chloroplastic free calcium in plants[J].Science,1995,269(5232):1863-1865.
[11] Urao T,Katagiri T,Mizoguchi T,et al.Two genes that encode Ca2+-depedent protein kinase are induced by drought and high salt stresses in Arebidopsis thaliana[J].Molecular Genetics and Genomics,1994,244(4):331-340.
[12] Camoni L,Fullone M R,Marra M,et al.The plasma membrane H-ATPase from maize roots in phosphorylated in the C-terminal domain by a calcium-dependent protein kinase[J].Physiology of Plant,1998,104(4):549-555.
[13] Huang J Z,Hardin S C,Huber S C.Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4[J].Archives of Bochemistry Biophysics,2001,393(1):61-66.
[14] Moutinho A,Trewavas A J,Malho R.Relocation of a Ca2+-dependent protein kinase activity during pollentube reorientation[J].Plant Cell,1998,10(9):1499-1509.
[15] Grabski S,Arnoys E,Busch B,et al.Regulation of actin tension in plant cells by kinases and phosphatases[J].Plant Physiology,1998,116(1):279-290.
[16] Frattini M,Morello L,Breviario D.Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development[J].Plant Molecular Biology,1999,41(6):753-764.
[17] 陈明耀.生物饵料培养[M].北京:中国农业出版社,1995:62-63.
[18] Saijo Y,Hata S,Kyozuka J,et al.Over-expression of a single Ca2+-dependent protein kinase confer both cold and salt/drought tolerance on rice plants[J].Plant Journal,2000,23(3):319-327.
[19] Kiselev K V, Grishchenko O V,Zhuravlev Y N.CDPK gene expression in salt tolerant rolB and rolC transformed cell cultures of Panax ginseng[J].Biologia Plantarum,2010,54(4):621-630.
[20] Romeis T,Ludwig A A,Martin R,et al.Calcium-dependent protein kinase plays an essential role in a plant defence response[J].Embo Journal,2001(20):5556-5567.
[21] Yuasa T,Okazaki Y,Iwasaki N,et al.Involvement of a calcium-dependent protein kinase in Hypoosmotic Turgor Regulationina Brackish Water Characeae Lamprothamnium succinctum[J].Plant & Cell Physiology,1997,38(5):586-594.
[22] Harmon A C,Cribskov M,Harper J F.CDPKs-B kinase for every Ca2+ signal[J].Trends in Plant Science, 2000,5(4):154-159.
[23] 张晓琳,柴晓杰,张婷,等.盐藻CDPK基因的克隆与生物信息学分析[J].核农学报,2013,27(4):418-424.
[24] Li Y.Recombinant production of antimicrobial peptides in Escherichia coli: a review[J].Protein Expression and Purification,2011,80(2):260-267.
[25] 王鸿鹤,朱孝峰,谢冰芬.人细胞周期蛋白D1及细胞周期蛋白激酶CDK4的原核表达及鉴定[J].中山大学学报,2003,24(5):444-447.
[26] Mueller R B,Pical C.Inositol phospholipid metabolism in arabidopsis,characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C[J].Plant Physiology,2002,130(1):22-46.
[27] Yuasa T,Muto S.Activation of a 40-kDa protein kinase in response to hypo-and hyperosmotic shock in the halotolerant green alga Dunaliella tertiolecta[J].Plant and Cell Physiology,1996,37(1):35-42.
[28] Kamigaki A,Mano S,Terauchi K,et al.Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor[J].The Plant Journal,2003,33(1):161-175.
[29] Durban M A, Silbersack J,Schweder T,et al.High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis[J].Applied Microbiology & Biotechnology,2007,74(3):634-639.
[2] Chen H,Lao Y M,Jiang J G.Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina[J].Science of The Total Environment,2011,409(7):1291-1297.
[3] Zhao L N,Gong W F,Chen X W,et al.Characterization of genes and enzymes in Dunaliella salina involved in glycerol metabolism in response to salt changes[J].Phycological Research,2013,61(1):37-45.
[4] He Q,Qiao D,Bai L,et al.Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina[J].Journal of Plant Physiology,2007,164(2):214-220.
[5] Jia Y,Xue L,Liu H,et al.Characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from the halotolerant alga Dunaliella salina and inhibition of its expression by RNAi[J].Current Microbiology,2009,58(5):426-431.
[6] Liska A J,Shevchenko A,Pick U,et al.Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics[J].Plant Physiology,2004,136(1):2806-2817.
[7] Katz A,Waridel P,Shevchenko A,et al.Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis[J].Molecular & Cellular Proteomics,2007,6(9):1459-1472.
[8] 陈硕,陈珈.植物中钙依赖蛋白激酶(CDPKs)的结构与功能[J].植物学通报,2001,18(2):143-148.
[9] Roberts D M,Harmon A C.Calcium-dependent proteins:target of intracellular calcium signals in higher plants[J].Annual Review of Plant Biology,1992(43):375-414.
[10] Johnson C H,Knight M R,Kondo T,et al.Circadian oscillations of cytosolic and chloroplastic free calcium in plants[J].Science,1995,269(5232):1863-1865.
[11] Urao T,Katagiri T,Mizoguchi T,et al.Two genes that encode Ca2+-depedent protein kinase are induced by drought and high salt stresses in Arebidopsis thaliana[J].Molecular Genetics and Genomics,1994,244(4):331-340.
[12] Camoni L,Fullone M R,Marra M,et al.The plasma membrane H-ATPase from maize roots in phosphorylated in the C-terminal domain by a calcium-dependent protein kinase[J].Physiology of Plant,1998,104(4):549-555.
[13] Huang J Z,Hardin S C,Huber S C.Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4[J].Archives of Bochemistry Biophysics,2001,393(1):61-66.
[14] Moutinho A,Trewavas A J,Malho R.Relocation of a Ca2+-dependent protein kinase activity during pollentube reorientation[J].Plant Cell,1998,10(9):1499-1509.
[15] Grabski S,Arnoys E,Busch B,et al.Regulation of actin tension in plant cells by kinases and phosphatases[J].Plant Physiology,1998,116(1):279-290.
[16] Frattini M,Morello L,Breviario D.Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development[J].Plant Molecular Biology,1999,41(6):753-764.
[17] 陈明耀.生物饵料培养[M].北京:中国农业出版社,1995:62-63.
[18] Saijo Y,Hata S,Kyozuka J,et al.Over-expression of a single Ca2+-dependent protein kinase confer both cold and salt/drought tolerance on rice plants[J].Plant Journal,2000,23(3):319-327.
[19] Kiselev K V, Grishchenko O V,Zhuravlev Y N.CDPK gene expression in salt tolerant rolB and rolC transformed cell cultures of Panax ginseng[J].Biologia Plantarum,2010,54(4):621-630.
[20] Romeis T,Ludwig A A,Martin R,et al.Calcium-dependent protein kinase plays an essential role in a plant defence response[J].Embo Journal,2001(20):5556-5567.
[21] Yuasa T,Okazaki Y,Iwasaki N,et al.Involvement of a calcium-dependent protein kinase in Hypoosmotic Turgor Regulationina Brackish Water Characeae Lamprothamnium succinctum[J].Plant & Cell Physiology,1997,38(5):586-594.
[22] Harmon A C,Cribskov M,Harper J F.CDPKs-B kinase for every Ca2+ signal[J].Trends in Plant Science, 2000,5(4):154-159.
[23] 张晓琳,柴晓杰,张婷,等.盐藻CDPK基因的克隆与生物信息学分析[J].核农学报,2013,27(4):418-424.
[24] Li Y.Recombinant production of antimicrobial peptides in Escherichia coli: a review[J].Protein Expression and Purification,2011,80(2):260-267.
[25] 王鸿鹤,朱孝峰,谢冰芬.人细胞周期蛋白D1及细胞周期蛋白激酶CDK4的原核表达及鉴定[J].中山大学学报,2003,24(5):444-447.
[26] Mueller R B,Pical C.Inositol phospholipid metabolism in arabidopsis,characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C[J].Plant Physiology,2002,130(1):22-46.
[27] Yuasa T,Muto S.Activation of a 40-kDa protein kinase in response to hypo-and hyperosmotic shock in the halotolerant green alga Dunaliella tertiolecta[J].Plant and Cell Physiology,1996,37(1):35-42.
[28] Kamigaki A,Mano S,Terauchi K,et al.Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor[J].The Plant Journal,2003,33(1):161-175.
[29] Durban M A, Silbersack J,Schweder T,et al.High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis[J].Applied Microbiology & Biotechnology,2007,74(3):634-639.