Isolation and Ectopic Expression of BoGPIAP from Bambusa oldhamii

DONG Li-li; SUN Hua-yu; ZHAO Han-sheng; LOU Yong-feng; WANG Li-li; GAO Zhi-min

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Isolation and Ectopic Expression of BoGPIAP from Bambusa oldhamii

1.
International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102

Received Date: 2014-07-31

Abstract

Glycosylphosphatidylinositol-anchored protein (GPIAP) plays an important role in many biological processes because of its diversity of structure and function. A GPIAP gene was isolated from Bambusa oldhamii using homologous cloning method, and designed as BoGPIAP. The full-length cDNA of BoGPIAP was 1772 bp including an open reading frame of 1 356 bp. The predicted protein encoded by BoGPIAP was 451 amino acids with a transmembrane structure at the N-end and a signal of GPI-anchor at the C-end, and also containing a typical conserved domain (47-211) and a CCVS motif, which indicated that it was a membrane protein belonging to GPIAP family. The plant expression vector with BoGPIAP::GFP was constructed and transformed into onion epidermal cells. The results of fluorescent microscope showed that the fused proteins were mainly located on the cytoplasmic membrane, which revealed that the protein encoded by BoGPIAP was a membrane protein. The expression vectors of sense and antisense BoGPIAP were constructed into the multiple cloning sites of pBI121 respectively, and transformed into tobacco mediated with agrobacterium. The transgenic plants of BoGPIAP were confirmed by PCR method. The phenotypes showed the antisense transgenic plants were thin, while the sense transgenic ones were stout comparing with the wild type. The average thickness of fibrous cell wall in the antisense transgenic plants was thinner, while that of the sense transgenic ones was significantly thicker than that of wild type, indicating that the BoGPIAP may play a regulatory role in the wall development of fibrous cell in bamboo.
- Bambusa oldhamii,
- glycosylphosphatidylinositol-anchored protein gene,
- subcellular localization,
- ectopic expression,
- fibrous cell

References

[1]	李雪平,高志民,彭镇华,等. 绿竹咖啡酸-O-甲基转移酶基因(COMT)的克隆及相关分析[J].林业科学研究,2007, 20(5):722-725.
[2]	金顺玉,卢孟柱,高健. 毛竹木质素合成相关基因C4H的克隆及组织表达分析[J]. 林业科学研究,2010, 23(3):319-325.
[3]	Hsieh L S, Yeh C S, Pan H C, et al. Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris[J]. Protein Expr Purif, 2010, 71(2):224-30.
[4]	杨学文,彭镇华. 一个毛竹细胞色素P450基因的克隆与表达研究[J]. 安徽农业大学学报,2010, 37(1):116-121.
[5]	Gao Z M, Wang X C, Peng Z H, et al. Characterization and primary functional analysis of phenylalanine ammonia-lyase gene from Phyllostachys edulis[J]. Plant Cell Rep, 2012, 31(7):1345-1356.
[6]	Hsieh L S, Hsieh Y L, Yeh C S, et al. Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii[J]. Mol Biol Rep, 2011, 38(1):283-290.
[7]	Hsieh L S, Yeh C S, Pan H C, et al. Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris[J]. Protein Expr Purif, 2010, 71(2):224-30.
[8]	杜亮亮,鲁专,金爱武. 雷竹纤维素合成酶基因cDNA克隆与表达分析[J]. 江西农业大学学报,2010, 32(3):535-540.
[9]	Chiu W B, Lin C H, Chang C J, et al. Molecular characterization and expression of four cDNAs encoding sucrose synthase from green bamboo Bambusa oldhamii[J]. New Phytol, 2006, 170(1):53-63.
[10]	高志民,杨学文,彭镇华,等.绿竹BoSUT2基因的克隆、分析与亚细胞定位研究[J]. 林业科学,2010, 46(2):45-49.
[11]	Roudier F, Fernandez A G, Fujita M, et al. COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation[J]. Plant Cell, 2005, 17(6):1749-1763.
[12]	Wasteneys G O, Fujita M. Establishing and maintaining axial growth:wall mechanical properties and the cytoskeleton[J]. J Plant Res, 2006, 119(1):5-10.
[13]	Sindhu A, Langewisch T, Olek A, et al. Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity[J]. Plant Physiol, 2007, 145(4):1444-1459.
[14]	Xiang J J, Zhang G H, Qian Q, et al. SEMI-ROLLEDLEAF1 encodes a putative glycosylphosphatidylinositol-anchored protein and modulates rice leaf rolling by regulating the formation of bulliform cells[J]. Plant Physiol, 2012, 159(4):1488-1500.
[15]	Li Y, Qian Q, Zhou Y, et al.BRITTLE CULM1, which encodes a COBRA-Like protein, affects the mechanical properties of rice plants[J]. Plant Cell, 2003, 15(9):2020-2031.
[16]	Liao S C, Lin C S, Wang A Y, et al. Differential expression of genes encoding acid invertases in multiple shoots of bamboo in response to various phytohormones and environmental factors[J]. J Agric Food Chem. 2013, 61(18):4396-405.
[17]	Yeh S H, Lee B H, Liao S C, et al. Identification of genes differentially expressed during the growth of Bambusa oldhamii[J]. Plant Physiol Biochem. 2013, 63:217-226.
[18]	Chen C Y, Hsieh M H, Yang C C, et al. Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii[J]. Phytochemistry, 2010, 71(11-12):1270-1279.
[19]	朱龙飞,徐英武,林新春. 绿竹花发育相关基因BoAP3的克隆与分析[J]. 浙江农林大学学报,2013, 30(6):839-842.
[20]	Lin X C, Huang L C, Fang W, et al. Understanding bamboo flowering based on large scale analysis of expressed sequence tags[J]. Gen Mol Res, 2010, 9(2):1085-1093.
[21]	Gao Z M, Li X P, Li L B, et al. An effective method for total RNA isolation from bamboo[J]. Chinese For Sci Tech, 2006, 5(3):52-54.
[22]	Kyte J, Doolittle R F. A simple method for displaying the hydropathic character of a protein[J]. J Mol Biol, 1982, 157, 105-132.
[23]	Sigrist C J, Cerutti L, de Castro E, et al. PROSITE, a protein domain database for functional characterization and annotation[J]. Nucleic Acids Res, 2010,38(Database issue):D161-6.
[24]	Geourjon C, Deléage G. SOPMA:Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Comput Appl Biosci, 1995, 11(6), 681-684.
[25]	Hofmann K, Stoffel W. TMbase-A database of membrane spanning proteins segments[J]. Biol Chem, 1993, Hoppe-Seyler 374, 166.
[26]	Petersen T N, Brunak S, von Heijne G, et al. SignalP 4.0:discriminating signal peptides from transmembrane regions[J]. Nat Methods, 2011, 8(10):785-786.
[27]	Gao Z M, Zheng B, Wang W Y, et al. Cloning and functional characterization of a GNA-like lectin from Chinese Narcissus (Narcissus tazetta var. Chinensis Roem)[J]. Physiol Plantarum, 2011,142(2):193-204.
[28]	徐刚标,卢孟柱,陆燕. ARF基因导人烟草的遗传转化研究[J]. 中南林业科技大学学报,2007, 27(5):6-12.
[29]	曹双平.植物单根纤维拉伸性能测试与评价[D]. 中国林业科学研究院硕士论文,2010.
[30]	Eisenhaber B, Bork P, Eisenhaber F. Sequence properties of GPI-anchored proteins near the omega-site:constraints for the polypeptide binding site of the putative transamidase[J]. Protein Eng, 1998, 11(12):1155-1161.
[31]	Eisenhaber B, Bork P, Eisenhaber F. Prediction of potential GPI-modification sites in proprotein sequences[J]. J Mol Biol, 1999, 292(3):741-758.
[32]	Udenfriend S, Kodukula K. How glycosylphosphatidylinositol-anchored membrane proteins are made[J]. Annu Rev Biochem, 1995, 64:563-591.
[33]	刘兵. 水稻GPI锚定蛋白基因OsGPA1的生物学功能研究[D]. 中山大学博士学位论文,2009.
[34]	Brady S M, Song S, Dhugga K S, et al. Combining expression and comparative evolutionary analysis. The COBRA gene family[J]. Plant Physiol, 2007, 143(1):172-187.
[35]	Sedbrook J C, Carroll K L, Hung K F, et al. The ArabidopsisSKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotien involved in directional root growth[J]. Plant Cell, 2002, 14(7):1635-1648.
[36]	Shi H, Kim Y, Guo Y, et al. The ArabidopsisSOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion[J]. Plant Cell, 2003, 15(1):19-32.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Isolation and Ectopic Expression of BoGPIAP from Bambusa oldhamii

1. International Center for Bamboo and Rattan, Key Laboratory on the Science and Technology of Bamboo and Rattan, Beijing 100102

Received Date: 2014-07-31

Abstract: Glycosylphosphatidylinositol-anchored protein (GPIAP) plays an important role in many biological processes because of its diversity of structure and function. A GPIAP gene was isolated from Bambusa oldhamii using homologous cloning method, and designed as BoGPIAP. The full-length cDNA of BoGPIAP was 1772 bp including an open reading frame of 1 356 bp. The predicted protein encoded by BoGPIAP was 451 amino acids with a transmembrane structure at the N-end and a signal of GPI-anchor at the C-end, and also containing a typical conserved domain (47-211) and a CCVS motif, which indicated that it was a membrane protein belonging to GPIAP family. The plant expression vector with BoGPIAP::GFP was constructed and transformed into onion epidermal cells. The results of fluorescent microscope showed that the fused proteins were mainly located on the cytoplasmic membrane, which revealed that the protein encoded by BoGPIAP was a membrane protein. The expression vectors of sense and antisense BoGPIAP were constructed into the multiple cloning sites of pBI121 respectively, and transformed into tobacco mediated with agrobacterium. The transgenic plants of BoGPIAP were confirmed by PCR method. The phenotypes showed the antisense transgenic plants were thin, while the sense transgenic ones were stout comparing with the wild type. The average thickness of fibrous cell wall in the antisense transgenic plants was thinner, while that of the sense transgenic ones was significantly thicker than that of wild type, indicating that the BoGPIAP may play a regulatory role in the wall development of fibrous cell in bamboo.

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Reference (36)

[1]	李雪平,高志民,彭镇华,等. 绿竹咖啡酸-O-甲基转移酶基因(COMT)的克隆及相关分析[J].林业科学研究,2007, 20(5):722-725.
[2]	金顺玉,卢孟柱,高健. 毛竹木质素合成相关基因C4H的克隆及组织表达分析[J]. 林业科学研究,2010, 23(3):319-325.
[3]	Hsieh L S, Yeh C S, Pan H C, et al. Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris[J]. Protein Expr Purif, 2010, 71(2):224-30.
[4]	杨学文,彭镇华. 一个毛竹细胞色素P450基因的克隆与表达研究[J]. 安徽农业大学学报,2010, 37(1):116-121.
[5]	Gao Z M, Wang X C, Peng Z H, et al. Characterization and primary functional analysis of phenylalanine ammonia-lyase gene from Phyllostachys edulis[J]. Plant Cell Rep, 2012, 31(7):1345-1356.
[6]	Hsieh L S, Hsieh Y L, Yeh C S, et al. Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii[J]. Mol Biol Rep, 2011, 38(1):283-290.
[7]	Hsieh L S, Yeh C S, Pan H C, et al. Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris[J]. Protein Expr Purif, 2010, 71(2):224-30.
[8]	杜亮亮,鲁专,金爱武. 雷竹纤维素合成酶基因cDNA克隆与表达分析[J]. 江西农业大学学报,2010, 32(3):535-540.
[9]	Chiu W B, Lin C H, Chang C J, et al. Molecular characterization and expression of four cDNAs encoding sucrose synthase from green bamboo Bambusa oldhamii[J]. New Phytol, 2006, 170(1):53-63.
[10]	高志民,杨学文,彭镇华,等.绿竹BoSUT2基因的克隆、分析与亚细胞定位研究[J]. 林业科学,2010, 46(2):45-49.
[11]	Roudier F, Fernandez A G, Fujita M, et al. COBRA, an Arabidopsis extracellular glycosyl-phosphatidyl inositol-anchored protein, specifically controls highly anisotropic expansion through its involvement in cellulose microfibril orientation[J]. Plant Cell, 2005, 17(6):1749-1763.
[12]	Wasteneys G O, Fujita M. Establishing and maintaining axial growth:wall mechanical properties and the cytoskeleton[J]. J Plant Res, 2006, 119(1):5-10.
[13]	Sindhu A, Langewisch T, Olek A, et al. Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity[J]. Plant Physiol, 2007, 145(4):1444-1459.
[14]	Xiang J J, Zhang G H, Qian Q, et al. SEMI-ROLLEDLEAF1 encodes a putative glycosylphosphatidylinositol-anchored protein and modulates rice leaf rolling by regulating the formation of bulliform cells[J]. Plant Physiol, 2012, 159(4):1488-1500.
[15]	Li Y, Qian Q, Zhou Y, et al.BRITTLE CULM1, which encodes a COBRA-Like protein, affects the mechanical properties of rice plants[J]. Plant Cell, 2003, 15(9):2020-2031.
[16]	Liao S C, Lin C S, Wang A Y, et al. Differential expression of genes encoding acid invertases in multiple shoots of bamboo in response to various phytohormones and environmental factors[J]. J Agric Food Chem. 2013, 61(18):4396-405.
[17]	Yeh S H, Lee B H, Liao S C, et al. Identification of genes differentially expressed during the growth of Bambusa oldhamii[J]. Plant Physiol Biochem. 2013, 63:217-226.
[18]	Chen C Y, Hsieh M H, Yang C C, et al. Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii[J]. Phytochemistry, 2010, 71(11-12):1270-1279.
[19]	朱龙飞,徐英武,林新春. 绿竹花发育相关基因BoAP3的克隆与分析[J]. 浙江农林大学学报,2013, 30(6):839-842.
[20]	Lin X C, Huang L C, Fang W, et al. Understanding bamboo flowering based on large scale analysis of expressed sequence tags[J]. Gen Mol Res, 2010, 9(2):1085-1093.
[21]	Gao Z M, Li X P, Li L B, et al. An effective method for total RNA isolation from bamboo[J]. Chinese For Sci Tech, 2006, 5(3):52-54.
[22]	Kyte J, Doolittle R F. A simple method for displaying the hydropathic character of a protein[J]. J Mol Biol, 1982, 157, 105-132.
[23]	Sigrist C J, Cerutti L, de Castro E, et al. PROSITE, a protein domain database for functional characterization and annotation[J]. Nucleic Acids Res, 2010,38(Database issue):D161-6.
[24]	Geourjon C, Deléage G. SOPMA:Significant improvement in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Comput Appl Biosci, 1995, 11(6), 681-684.
[25]	Hofmann K, Stoffel W. TMbase-A database of membrane spanning proteins segments[J]. Biol Chem, 1993, Hoppe-Seyler 374, 166.
[26]	Petersen T N, Brunak S, von Heijne G, et al. SignalP 4.0:discriminating signal peptides from transmembrane regions[J]. Nat Methods, 2011, 8(10):785-786.
[27]	Gao Z M, Zheng B, Wang W Y, et al. Cloning and functional characterization of a GNA-like lectin from Chinese Narcissus (Narcissus tazetta var. Chinensis Roem)[J]. Physiol Plantarum, 2011,142(2):193-204.
[28]	徐刚标,卢孟柱,陆燕. ARF基因导人烟草的遗传转化研究[J]. 中南林业科技大学学报,2007, 27(5):6-12.
[29]	曹双平.植物单根纤维拉伸性能测试与评价[D]. 中国林业科学研究院硕士论文,2010.
[30]	Eisenhaber B, Bork P, Eisenhaber F. Sequence properties of GPI-anchored proteins near the omega-site:constraints for the polypeptide binding site of the putative transamidase[J]. Protein Eng, 1998, 11(12):1155-1161.
[31]	Eisenhaber B, Bork P, Eisenhaber F. Prediction of potential GPI-modification sites in proprotein sequences[J]. J Mol Biol, 1999, 292(3):741-758.
[32]	Udenfriend S, Kodukula K. How glycosylphosphatidylinositol-anchored membrane proteins are made[J]. Annu Rev Biochem, 1995, 64:563-591.
[33]	刘兵. 水稻GPI锚定蛋白基因OsGPA1的生物学功能研究[D]. 中山大学博士学位论文,2009.
[34]	Brady S M, Song S, Dhugga K S, et al. Combining expression and comparative evolutionary analysis. The COBRA gene family[J]. Plant Physiol, 2007, 143(1):172-187.
[35]	Sedbrook J C, Carroll K L, Hung K F, et al. The ArabidopsisSKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotien involved in directional root growth[J]. Plant Cell, 2002, 14(7):1635-1648.
[36]	Shi H, Kim Y, Guo Y, et al. The ArabidopsisSOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion[J]. Plant Cell, 2003, 15(1):19-32.

Isolation and Ectopic Expression of BoGPIAP from Bambusa oldhamii

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通讯作者: 陈斌, bchen63@163.com

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Isolation and Ectopic Expression of BoGPIAP from Bambusa oldhamii

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