桂糖11号Rubisco基因克隆、原核表达及纯化
|
摘要
【目的】通过原核表达及纯化获得甘蔗1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)的大、小亚基融合蛋白,为获得符合抗体制备条件的高纯度融合蛋白提供技术支持。【方法】以甘蔗品种桂糖11号(GT11)+1叶为材料,根据NCBI公布的甘蔗Rubisco大、小亚基基因(rbc L和rbc S)的编码域序列(CDS)设计特异性引物,PCR扩增rbc L和rbc S基因的CDS,然后连接至原核表达载体p ET-30a(+),构建重组质粒后转入原核细胞(大肠杆菌Rosetta)中诱导表达,用镍亲和层析柱对融合蛋白进行纯化,并比较分析桂糖11号与其他甘蔗品种在rbc L和rbc S基因的CDS及编码蛋白序列方面的差异。【结果】rbc L和rbc S基因的CDS长度分别为1431和510 bp,其中桂糖11号rbc L基因的CDS与Saccharum hybrid cultivar SP80-3280(Gen Bank登录号AE009947)、Saccharum hybrid cultivar Q155(Gen Bank登录号KU214867)的一致,桂糖11号rbc S基因的CDS与Saccharum hybrid cultivar GT28(Gen Bank登录号JN591757)的存在7个碱基差异,但仅有2个氨基酸发生错义突变。Rbc L和Rbc S融合蛋白以包涵体形式存在原核细胞中,用镍离子亲和层析纯化及超滤浓缩后其浓度均在1.0 mg/m L以上。【结论】从桂糖11号成功克隆Rubisco大亚基和小亚基基因的CDS,大亚基基因的CDS比小亚基的保守性更高,且均可在原核细胞中高度表达,经纯化浓缩获得的高纯度融合蛋白可用于制备Rubisco单克隆抗体。
【Objective】In order to technical reference for getting high-purity fusion proteins that met preparation conditions of monoclonal antibody, the large and small subunits of ribulose-1,5-bisphosphosphate carboxylase/oxygenase(Rubisco) from sugarcane were expressed in prokaryote, and then purified and concentrated. 【Method】Using leaf(+1) of sugarcane cultivar Guitang 11(GT11) as material, the specific primers of large and small subunit genes(rbc L and rbc S) of rubisco were designed based on coding domain sequences(CDSs) of rbc L and rbc S genes published on the NCBI. Then the rbc L and rbc S genes were amplified by PCR, and inserted to prokaryotic expression vector p ET-30a(+) to construct recombinant plasmids, respectively. The recombinant plasmids were transferred into prokaryotic cell(Escherichia coli Rosetta), and the over-expressed fusion proteins were purified with Ni affinity chromatography. Meanwhile, the CDSs of nucleotide and amino acid of rbc S and rbc L genes between GT11 and other sugarcane cultivars were analyzed and compared.【Result】The CDSs of rbc L and rbc S genes were 1431 and 510 bp in length, respectively. The CDS of GT11 rbc L gene was identical to those of Saccharum hybrid cultivar SP-80-3280(Gen Bank: AE009947.2) and Saccharum hybrid cultivar Q155(Gen Bank: KU214867.1), while CDS of GT11 rbc S gene was different in seven nucleotides from Saccharum hybrid cultivar GT28(Gen Bank: JN591757.1), only leading to two missense mutation. The Rbc L and Rbc S fusion proteins were expressed in the form of inclusion bodies in prokaryotic cell, and then purified with Ni affinity chromatography and concentrated to 1.0 mg/m L by ultrafiltration. 【Conclusion】The CDSs of rubisco rbc L and rbc S genes have been successfully cloned from sugarcane variety GT11. The rbc L gene CDS is highly conserved compared with rbc S gene CDS. Moreover, the rbc L and rbc S genes are over-expressed in the prokaryotic cell, and the high-purity fusion proteins are obtained successfully by purification and concentration, which can be used to prepare monoclonal antibodies.
【Objective】In order to technical reference for getting high-purity fusion proteins that met preparation conditions of monoclonal antibody, the large and small subunits of ribulose-1,5-bisphosphosphate carboxylase/oxygenase(Rubisco) from sugarcane were expressed in prokaryote, and then purified and concentrated. 【Method】Using leaf(+1) of sugarcane cultivar Guitang 11(GT11) as material, the specific primers of large and small subunit genes(rbc L and rbc S) of rubisco were designed based on coding domain sequences(CDSs) of rbc L and rbc S genes published on the NCBI. Then the rbc L and rbc S genes were amplified by PCR, and inserted to prokaryotic expression vector p ET-30a(+) to construct recombinant plasmids, respectively. The recombinant plasmids were transferred into prokaryotic cell(Escherichia coli Rosetta), and the over-expressed fusion proteins were purified with Ni affinity chromatography. Meanwhile, the CDSs of nucleotide and amino acid of rbc S and rbc L genes between GT11 and other sugarcane cultivars were analyzed and compared.【Result】The CDSs of rbc L and rbc S genes were 1431 and 510 bp in length, respectively. The CDS of GT11 rbc L gene was identical to those of Saccharum hybrid cultivar SP-80-3280(Gen Bank: AE009947.2) and Saccharum hybrid cultivar Q155(Gen Bank: KU214867.1), while CDS of GT11 rbc S gene was different in seven nucleotides from Saccharum hybrid cultivar GT28(Gen Bank: JN591757.1), only leading to two missense mutation. The Rbc L and Rbc S fusion proteins were expressed in the form of inclusion bodies in prokaryotic cell, and then purified with Ni affinity chromatography and concentrated to 1.0 mg/m L by ultrafiltration. 【Conclusion】The CDSs of rubisco rbc L and rbc S genes have been successfully cloned from sugarcane variety GT11. The rbc L gene CDS is highly conserved compared with rbc S gene CDS. Moreover, the rbc L and rbc S genes are over-expressed in the prokaryotic cell, and the high-purity fusion proteins are obtained successfully by purification and concentration, which can be used to prepare monoclonal antibodies.
引文
曹凯鸣,袁卫明,詹树萱,顾其敏,徐豹.1996.野生大豆rbc S基因的克隆及结构分析[J].植物学报,38(9):753-756.Cao K M,Yuan W M,Zhan S X,Gu Q M,Xu B.1996.Cloning and structure analysis of rbc S gene from wild soybean[J].Acta Botanica Sinica,38(9):753-756.
崔喜艳,董雅致,刘晓庆,张治安.2014.栽培大豆Rubisco小亚基基因(rbc S)全长c DNA的克隆及原核表达[J].中国油料作物学报,36(3):329-333.Cui X Y,Dong Y Z,Liu X Q,Zhang Z A.2014.Cloning and prokaryotic expression of full-length c DNA of rubisco small subunit gene(rbc S)in Glyine ma[J].Chinese Journal of Oil Crop Sciences,36(3):329-333.
贺超英,王伟权,东方阳,张劲松,盖钧镒,陈受宜.2001.大豆1,5-二磷酸核酮糖羧化酶小亚基基因的转录表达分析[J].科学通报,46(16):1375-1380.He C Y,Wang W Q,Dong F Y,Zhang J S,Gai J Y,Chen S Y.2001.Transcription and expression analysis on small subunit gene of rubisco in soybean[J].Chinese Science Bulletin,46(16):1375-1380.
黄东亮,覃肖良,廖青,高轶静,方锋学.2010.高质量甘蔗基因组DNA的简便快速提取方法研究[J].生物技术通报,(5):101-106.Huang D L,Qin X L,Liao Q,Gao Y J,Fang F X.2010.Simple and rapid procedure for isolation of high quality genomic DNA from sugarcane[J].Biotechnology Bulletin,(5):101-106.
黄瑶,马诚.1994.叶绿体DNA及其在植物系统学研究中的应用[J].植物学通报,11(2):11-25.Huang Y,Ma C.1994.Chloroplast DNA and its application to plant systematic studies[J].Chinese Bulletin of Botany,11(2):11-25.
李凤玲,吴光耀.1996.Rubisco的研究进展[J].生物学通报,31(3):8-9.Li F L,Wu G Y.1996.Research progress of rubisco[J].Bulletin of Biology,31(3):8-9.
李杨瑞,杨丽涛.2009.20世纪90年代以来我国甘蔗产业和科技的新发展[J].西南农业学报,22(5):1469-1476.Li Y R,Yang L T.2009.New developments in sugarcane industry and technologies in China since 1990[J].Southwest China Journal of Agricultural Sciences,22(5):1469-1476.
刘晓庆,崔喜艳,丁志鑫,李海燕.2011.大豆rbc L基因克隆、序列分析及原核表达[J].中国油料作物学报,33(3):226-230.Liu X Q,Cui X Y,Ding Z X,Li H Y.2011.Cloning,sequence analysis and prokaryotic expression of rbc L gene from Glycine max[J].Chinese Journal of Oil Crop Sciences,33(3):226-230.
谢翎,陈红梅,尹翀,项小燕,黄勃.2011.大肠杆菌DH5α菌株感受态制备及转化率变化研究[J].生物学杂志,28(4):4-6.Xie L,Chen H M,Yin C,Xiang X Y,Huang B.2011.Study on variation of transform action rate of E.coli DH5αcompetent cells[J].Journal of Biology,28(4):4-6.
Andersson I,Backlund A.2008.Structure and function of rubisco[J].Plant Physiology&Biochemistry Ppb,46(3):275-291.
Andersson I,Knight S,Schneider G,Schneider G,Lindqvist Y,Lundqvist T,Branden C I,Lorimer G H.1989.Crystal structure of the active site of ribulose-bisphosphate carboxylase[J].Nature,337:229-234.
Bedbrook J R,Smith S M,Ellis R J.1980.Molecular cloning and sequencing of c DNA encoding the precursor to the small subunit of chloroplast ribulose-1,5-bisphosphate carboxylase[J].Nature,287:692-697.
Coen D M,Bedbrook J R,Bogorad L,Rich A.1977.Maize chloroplast DNA fragment encoding large subunit of ribulose bisphosphate carboxylase[J].Proceedings of the National Academy of Sciences,74(12):5487-5491.
Gontcharov A A,Marin B,Melkonian M.2004.Are combined analyses better than single gene phylogenies?A case study using SSU r DNA and rbc L sequence comparisons in the Zygnematophyceae(Streptophyta)[J].Molecular and Biological Evolution,21(3):612-624.
Gurevitz M,Somerville C R,Mcintosh L.1985.Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia col[iJ].Proceedings of the National Academy of Sciences,82(19):6546-6550.
Li Y R,Yang L T.2015.Sugarcane agriculture and sugar industry in China[J].Sugar Tech,17(1):1-8.
Sasanuma T.2001.Characterization of the rbc S multigene family in wheat:subfamily classification,determination of chromosomal location and evolutionary analysis[J].Molecular Genetics&Genomics,265(1):161-171.
Thomas-Hall S,Campbell P R,Carlens K,Kawanishi E,Swennen R,Sági L,Schenk P M.2007.Phylogenetic and molecular analysis of the ribulose-1,5-bisphosphate carboxylase small subunit gene family in banana[J].Journal of Experimental Botany,58(10):2685-2697.
Valentin K,Zetsche K.1989.The genes of both subunits of ribulose-1,5-bisphosphate carboxylase constitute an operon on the plastome of a red alga[J].Current Genetics,16(3):203-209.
Wildman S G,Bonner J.1947.The proteins of green leaves;isolation,enzymatic properties and auxin content of spinach cytoplasmic proteins[J].Archives of Biochemistry,14(3):381-413.
Yamazaki T,Yamamoto M,Sakamoto W,Kawano S.2005.Isolation and molecular characterization of rbc S in the unicellular green alga Nannochloris bacillaris(Chlorophyta,Trebouxiophyceae)[J].Phycological Research,53(1):67-76.
崔喜艳,董雅致,刘晓庆,张治安.2014.栽培大豆Rubisco小亚基基因(rbc S)全长c DNA的克隆及原核表达[J].中国油料作物学报,36(3):329-333.Cui X Y,Dong Y Z,Liu X Q,Zhang Z A.2014.Cloning and prokaryotic expression of full-length c DNA of rubisco small subunit gene(rbc S)in Glyine ma[J].Chinese Journal of Oil Crop Sciences,36(3):329-333.
贺超英,王伟权,东方阳,张劲松,盖钧镒,陈受宜.2001.大豆1,5-二磷酸核酮糖羧化酶小亚基基因的转录表达分析[J].科学通报,46(16):1375-1380.He C Y,Wang W Q,Dong F Y,Zhang J S,Gai J Y,Chen S Y.2001.Transcription and expression analysis on small subunit gene of rubisco in soybean[J].Chinese Science Bulletin,46(16):1375-1380.
黄东亮,覃肖良,廖青,高轶静,方锋学.2010.高质量甘蔗基因组DNA的简便快速提取方法研究[J].生物技术通报,(5):101-106.Huang D L,Qin X L,Liao Q,Gao Y J,Fang F X.2010.Simple and rapid procedure for isolation of high quality genomic DNA from sugarcane[J].Biotechnology Bulletin,(5):101-106.
黄瑶,马诚.1994.叶绿体DNA及其在植物系统学研究中的应用[J].植物学通报,11(2):11-25.Huang Y,Ma C.1994.Chloroplast DNA and its application to plant systematic studies[J].Chinese Bulletin of Botany,11(2):11-25.
李凤玲,吴光耀.1996.Rubisco的研究进展[J].生物学通报,31(3):8-9.Li F L,Wu G Y.1996.Research progress of rubisco[J].Bulletin of Biology,31(3):8-9.
李杨瑞,杨丽涛.2009.20世纪90年代以来我国甘蔗产业和科技的新发展[J].西南农业学报,22(5):1469-1476.Li Y R,Yang L T.2009.New developments in sugarcane industry and technologies in China since 1990[J].Southwest China Journal of Agricultural Sciences,22(5):1469-1476.
刘晓庆,崔喜艳,丁志鑫,李海燕.2011.大豆rbc L基因克隆、序列分析及原核表达[J].中国油料作物学报,33(3):226-230.Liu X Q,Cui X Y,Ding Z X,Li H Y.2011.Cloning,sequence analysis and prokaryotic expression of rbc L gene from Glycine max[J].Chinese Journal of Oil Crop Sciences,33(3):226-230.
谢翎,陈红梅,尹翀,项小燕,黄勃.2011.大肠杆菌DH5α菌株感受态制备及转化率变化研究[J].生物学杂志,28(4):4-6.Xie L,Chen H M,Yin C,Xiang X Y,Huang B.2011.Study on variation of transform action rate of E.coli DH5αcompetent cells[J].Journal of Biology,28(4):4-6.
Andersson I,Backlund A.2008.Structure and function of rubisco[J].Plant Physiology&Biochemistry Ppb,46(3):275-291.
Andersson I,Knight S,Schneider G,Schneider G,Lindqvist Y,Lundqvist T,Branden C I,Lorimer G H.1989.Crystal structure of the active site of ribulose-bisphosphate carboxylase[J].Nature,337:229-234.
Bedbrook J R,Smith S M,Ellis R J.1980.Molecular cloning and sequencing of c DNA encoding the precursor to the small subunit of chloroplast ribulose-1,5-bisphosphate carboxylase[J].Nature,287:692-697.
Coen D M,Bedbrook J R,Bogorad L,Rich A.1977.Maize chloroplast DNA fragment encoding large subunit of ribulose bisphosphate carboxylase[J].Proceedings of the National Academy of Sciences,74(12):5487-5491.
Gontcharov A A,Marin B,Melkonian M.2004.Are combined analyses better than single gene phylogenies?A case study using SSU r DNA and rbc L sequence comparisons in the Zygnematophyceae(Streptophyta)[J].Molecular and Biological Evolution,21(3):612-624.
Gurevitz M,Somerville C R,Mcintosh L.1985.Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia col[iJ].Proceedings of the National Academy of Sciences,82(19):6546-6550.
Li Y R,Yang L T.2015.Sugarcane agriculture and sugar industry in China[J].Sugar Tech,17(1):1-8.
Sasanuma T.2001.Characterization of the rbc S multigene family in wheat:subfamily classification,determination of chromosomal location and evolutionary analysis[J].Molecular Genetics&Genomics,265(1):161-171.
Thomas-Hall S,Campbell P R,Carlens K,Kawanishi E,Swennen R,Sági L,Schenk P M.2007.Phylogenetic and molecular analysis of the ribulose-1,5-bisphosphate carboxylase small subunit gene family in banana[J].Journal of Experimental Botany,58(10):2685-2697.
Valentin K,Zetsche K.1989.The genes of both subunits of ribulose-1,5-bisphosphate carboxylase constitute an operon on the plastome of a red alga[J].Current Genetics,16(3):203-209.
Wildman S G,Bonner J.1947.The proteins of green leaves;isolation,enzymatic properties and auxin content of spinach cytoplasmic proteins[J].Archives of Biochemistry,14(3):381-413.
Yamazaki T,Yamamoto M,Sakamoto W,Kawano S.2005.Isolation and molecular characterization of rbc S in the unicellular green alga Nannochloris bacillaris(Chlorophyta,Trebouxiophyceae)[J].Phycological Research,53(1):67-76.