Bioinformatics and Expression Pattern Analysis of Tomato ns LTP 2-like cDNA full-length Gene Clone
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摘要
TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.
TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.
TDF1(transcription-drived fragment) was homologous to the predicted S. lycopersicum nonspecific lipid-transfer protein,nsLTP 2-like(91%), and it was significantly upregulated in response to C. fulvum(cladosporium fulvum) infection in tomato plants.In this experiment, the full-length cDNA of nsLTP 2-like was cloned using RACE technology based on the sequence of TDF1(GenBank: JZ717725). A full-length, 625 bp(GenBank: KU366289), cDNA sequence, which with 98% similarity to nsLTP 2-like gene(GenBank: XM015233692) was obtained. This cDNA contains an ORF(open reading frame) with full-length of 345 bp, coding of 114 amino acids, including 12.3% Ala and Gly. Protein molecular weight was 11.51 ku, the isoelectric point(pI) was 8.99, and average overall hydrophilicity was 0.412, with one phosphorylation sites, belonging to volatile acidic nuclear protein. Secondary structure prediction showed that α-Helix accounts for 30.7%, extension chain for 12.28%, β-corner for 9.65%, and random coil for 47.37%. Through comparative analysis of the homology among species, it was found that the amino acid sequence of tomato nsLTP 2-like protein had a high similarity with other plants, and with a specific conserved sequence which might related features in nsLTP 2-like protein. It also be analyzed the gene expression pattern of tomato in different parts and under different stress conditions.The results showed that nsLTP 2-like gene was up-regulated in varying degrees, under the condition of cold stress, exogenous hormone spraying and cladosporium fulvum infection. Therefore, it was speculated that the gene played a role in response to abiotic and biotic stress in tomato.
引文
Blein J P,Coutos T P,Marion D,et al.2002.Fromelicitins to lipidtransfer proteins:a new insight in cell signaling involved in plant defence mechanisms.Trends Plant Science,7(7):293-296.
Boutrot F,Chantret N,Gautier M F.2008.Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein(nsLTP)gene families and identification of wheat nsLTP genes by EST data mining.BMC Genomics,9:86.
Boutrot F,Guirao A,Alary R,et al.2005.Wheat non-specific lipid transfer protein genes display a complex pattern of expression in developing seeds.Biochemical et Biophysica Acta,1730:114-125.
Chae K,Kieslich C A,Morikis D,et al.2009.A gain-of-function mutation of Arabidopsis lipid transfer protein five disturbs pollen tube tip growth and fertilization.The Plant Cell,21:3902-3914.
Chae K,Zhang K,Zhang L,et al.2007.Two SCA(stigma/style cysteine-rich adhesin)isoforms show structural differences that correlate with their levels of in vitro pollen tube adhesion activity.JBiol Chem,282:33845-33858.
Hincha D K,Neukamm B,Sror H A,et al.2001.Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family.Plant Physiology,125:835-846.
Kader J C.1996.Lipid transfer protein in plants.Ann Rev Plant Physiol Plant Mol Biol,47:627-654.
Kimberly D C,Mark A T,Lawrence B S.2006.Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco.Plant Physiology,140:176-183.
Lu Z X,Gaudet D,Puchalski B,et al.2005.Inducers of resistance reduce common bunt infection in wheat seedlings while differentially regulating defence-gene expression.Physiol Mol Plant Pathol,67(3):138-148.
Nielsen K K,Nielsen J E,Madrid S M.1996.New antifungal proteins from sugar beet(Beta vulgaris L.)showing homology to nonspecific lipid transfer proteins.Plant Mol Biol,31:539-552.
Pagnussat L A,Lombardo C,Regente M,et al.2009.Unexpected localization of a lipid transfer protein in germinating sunflower seeds.Journal of Plant Physiology,166:797-806.
Pii Y,Pandolfini T,Crimi M.2010.Signaling LTPs:a new plant LTPs sub-family.Plant Signal Behave,5(5):594-597.
Sawano Y,Hatano K,Miyakawa T.2008.Proteinase inhibitor from ginkgo seeds is a member of plant non-specific lipid transfer protein gene family.Plant Physiol,146(7):1409-1919.
Sossountzov L,Ruiz-Avila L,Vignols F,et al.1991.Spatial and temporal expression of a maize lipid transfer protein gene.The Plant Cell,3:923-933.
Thoma S,Hecht U,Kippers A,et al.1994.Tissue specific expression of a gene encoding a cell wall localized lipid transfer protein from Arabildopsis thaliana.Plant Physiology,105:35-45.
Verdoy D,Lucas M M,Manrique E,et al.2004.Differential organspecific response to salt stress and water deficit in nodulated bean(Phaseolus vulgaris).Plant Cell Environ,27(6):757-767.
Werner R B,Sharon T,Jose B,et al.1991.Isolation of a cDNAclone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway.Plant Physiology,95:164-170.
Yeats T H,Rose J K C.2008.The biochemistry and biology of extracellular plant lipid-transfer proteins(LTPs).Protein Sci,17:191-198.
Boutrot F,Chantret N,Gautier M F.2008.Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein(nsLTP)gene families and identification of wheat nsLTP genes by EST data mining.BMC Genomics,9:86.
Boutrot F,Guirao A,Alary R,et al.2005.Wheat non-specific lipid transfer protein genes display a complex pattern of expression in developing seeds.Biochemical et Biophysica Acta,1730:114-125.
Chae K,Kieslich C A,Morikis D,et al.2009.A gain-of-function mutation of Arabidopsis lipid transfer protein five disturbs pollen tube tip growth and fertilization.The Plant Cell,21:3902-3914.
Chae K,Zhang K,Zhang L,et al.2007.Two SCA(stigma/style cysteine-rich adhesin)isoforms show structural differences that correlate with their levels of in vitro pollen tube adhesion activity.JBiol Chem,282:33845-33858.
Hincha D K,Neukamm B,Sror H A,et al.2001.Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family.Plant Physiology,125:835-846.
Kader J C.1996.Lipid transfer protein in plants.Ann Rev Plant Physiol Plant Mol Biol,47:627-654.
Kimberly D C,Mark A T,Lawrence B S.2006.Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco.Plant Physiology,140:176-183.
Lu Z X,Gaudet D,Puchalski B,et al.2005.Inducers of resistance reduce common bunt infection in wheat seedlings while differentially regulating defence-gene expression.Physiol Mol Plant Pathol,67(3):138-148.
Nielsen K K,Nielsen J E,Madrid S M.1996.New antifungal proteins from sugar beet(Beta vulgaris L.)showing homology to nonspecific lipid transfer proteins.Plant Mol Biol,31:539-552.
Pagnussat L A,Lombardo C,Regente M,et al.2009.Unexpected localization of a lipid transfer protein in germinating sunflower seeds.Journal of Plant Physiology,166:797-806.
Pii Y,Pandolfini T,Crimi M.2010.Signaling LTPs:a new plant LTPs sub-family.Plant Signal Behave,5(5):594-597.
Sawano Y,Hatano K,Miyakawa T.2008.Proteinase inhibitor from ginkgo seeds is a member of plant non-specific lipid transfer protein gene family.Plant Physiol,146(7):1409-1919.
Sossountzov L,Ruiz-Avila L,Vignols F,et al.1991.Spatial and temporal expression of a maize lipid transfer protein gene.The Plant Cell,3:923-933.
Thoma S,Hecht U,Kippers A,et al.1994.Tissue specific expression of a gene encoding a cell wall localized lipid transfer protein from Arabildopsis thaliana.Plant Physiology,105:35-45.
Verdoy D,Lucas M M,Manrique E,et al.2004.Differential organspecific response to salt stress and water deficit in nodulated bean(Phaseolus vulgaris).Plant Cell Environ,27(6):757-767.
Werner R B,Sharon T,Jose B,et al.1991.Isolation of a cDNAclone for spinach lipid transfer protein and evidence that the protein is synthesized by the secretory pathway.Plant Physiology,95:164-170.
Yeats T H,Rose J K C.2008.The biochemistry and biology of extracellular plant lipid-transfer proteins(LTPs).Protein Sci,17:191-198.