棉花GhSP1L基因克隆与表达分析
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摘要
【目的】棉花Spiral1-LIKE(SP1L)基因(Gh SP1L)的克隆、功能序列分析及原核表达。【方法】通过RT-PCR从棉纤维中克隆Gh SP1L基因,进行Gh SP1L蛋白的功能结构域分析,构建原核表达载体p ET28a-GhSP1L,进行蛋白体外诱导表达,SDS-PAGE分析蛋白的表达情况;RT-PCR检测Gh SP1L基因的表达特征。【结果】从陆地棉纤维组织中克隆得到Gh SP1L基因的全长c DNA,其开放读码框为318 bp,编码106个氨基酸的蛋白质,理论分子质量为11.76 k Da。对氨基酸序列进行生物信息学分析,其N端和C端较为保守,含有SCOP结构域,属于SP1L家族蛋白,能够在其他微管蛋白协助下与微管结合,SPR1的定位可能与其结合的微管结合蛋白相关。构建了p ET28a-Gh SP1L原核表达载体,并通过体外诱导表达后获得分子质量约为12 k Da左右的重组蛋白Gh SP1L;半定量RT-PCR结果表明,Gh SP1L基因开花后3 d的纤维组织中表达量较高。【结论】Gh SP1L基因的克隆、序列分析和表达,重组蛋白的诱导表达,为深入研究该基因在棉纤维发育中的作用奠定了基础。
【Objective】Cloning a cotton Gh SP1 L gene from fiber tissue,analyzing sequence function structure domain and Gh SP1 L expression characteristics during the fiber development. 【Method 】Gh SP1 L gene was cloned from fiber tissue through RT- PCR method. A series of bioinformatics software were used to analyze sequence characteristics. The recombinant protein Gh SP1 L was obtained by transforming p ET28a- Gh SP1 L vector into E. Coli BL21( DE3) with in vitro induction,and SDS- PAGE analysis. Gh SP1 L gene expression analysis was performed by semi- quantitative RT- PCR method. 【Result】A cotton Gh SP1 L full- length c DNA was cloned from fiber tissue. Gh SP1 L c DNA contains a 318 bp open reading frame coding a ~ 12 k Da protein of 108 amino acids. Gh SP1 L protein contains several SCOP functional domains. Phylogenetic tree analysis showed that Gh SP1 L belonged to the SP1L1 protein family with the function of binding to microtubule through assistance of other microtubule protein,which inferred that the location of SPR1 was related with its binding microtubule protein. 12 k Da recombinant protein Gh SP1 L were obtained by SDS- PAGE analysis; Semi-quantitative RT- PCR analysis showed that the Gh SP1 L gene expressed highly at 3- dpa fiber tissue,and was closely related to the process of cotton fiber initiation development.【Conclusion】Gh SP1 L cloning,functional sequence analysis and expression analysis established a basis for its further functions in cotton fiber development.
【Objective】Cloning a cotton Gh SP1 L gene from fiber tissue,analyzing sequence function structure domain and Gh SP1 L expression characteristics during the fiber development. 【Method 】Gh SP1 L gene was cloned from fiber tissue through RT- PCR method. A series of bioinformatics software were used to analyze sequence characteristics. The recombinant protein Gh SP1 L was obtained by transforming p ET28a- Gh SP1 L vector into E. Coli BL21( DE3) with in vitro induction,and SDS- PAGE analysis. Gh SP1 L gene expression analysis was performed by semi- quantitative RT- PCR method. 【Result】A cotton Gh SP1 L full- length c DNA was cloned from fiber tissue. Gh SP1 L c DNA contains a 318 bp open reading frame coding a ~ 12 k Da protein of 108 amino acids. Gh SP1 L protein contains several SCOP functional domains. Phylogenetic tree analysis showed that Gh SP1 L belonged to the SP1L1 protein family with the function of binding to microtubule through assistance of other microtubule protein,which inferred that the location of SPR1 was related with its binding microtubule protein. 12 k Da recombinant protein Gh SP1 L were obtained by SDS- PAGE analysis; Semi-quantitative RT- PCR analysis showed that the Gh SP1 L gene expressed highly at 3- dpa fiber tissue,and was closely related to the process of cotton fiber initiation development.【Conclusion】Gh SP1 L cloning,functional sequence analysis and expression analysis established a basis for its further functions in cotton fiber development.
引文
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[11]Li,X.B.,Cai,L.,Cheng,N.H.,&Liu,J.W.(2002).Molecular characterization of the cotton Gh TUB1 gene that is preferentially expressed in fiber.Plant Physiology,130(2):666-674.
[12]蒋建雄,张天真.利用CTAB/酸酚法提取棉花组织总RNA[J].棉花学报,2003,15(3):166-167.JIANG Jian-xiong,ZHANG Tian-zhen.(2003).Extraction of Total RNA in Cotton Tissues with CTAB-acidic Phenolic Method[J].Cotton Science,15(3):166-167.(in Chinese)
[13]田大鹏,葛娟石峰,等.棉花Gh DHAR2基因的克隆、功能序列分析及原核表达[J].生物技术通报,2012,(7):65-69.TIAN Da-peng,GE Juan,SHI Feng,et al.(2012).Cloning,Functional Sequence Analysis and Prokaryotic Expression of Cotton Gh DHAR2 c DNA[J].Biotechnology Bulletin,(7):65-69.(in Chinese)
[14]李学宁,杜军伟,李鸿彬.棉花脱氢抗坏血酸还原酶基因的克隆、原核表达与纯化[J].石河子大学学报(自然科学版),2010,28(5):542-545.LI Xue-ning,DU Jun-wei,LI Hong-bin.(2010).Cloning,Prokaryotic Expression and Purification of a Cotton Dehydroascorbate Reductase Gene[J].Journal of Shihezi University(Natural Science),28(5):542-545.(in Chinese)
[15]王芳,王斐,孙辉,等.棉花Gh DHAR3基因克隆、功能序列分析及烟草的遗传转化[J].西北农业学报,2011,20(5):88-93.WANG Fang,WANG Fei,SUN Hui,et al.(2011).Cloning and Sequence Analysis of a Cotton Ch DHAR3 c DNA and Genetic Transformation of Tobacco[J].Acta Agriculturae Boreali-occidentalis Sinica,20(5):88-93.(in Chinese)
[16]Cyr,R.J.,&Palevitz,B.A.(1995).Organization of cortical microtubules in plant cells.Current opinion in cell biology,7(1):65-71.
[17]Lloyd,C.(1994).Why should stationary plant cells have such dynamic microtubules.Molecular biology of the cell,5(12):1,277.
[18]Gundersen,G.G.,&Cook,T.A.(1999).Microtubules and signal transduction.Current opinion in cell biology,11(1):81-94.
[19]Hashimoto,T.(2003).Dynamics and regulation of plant interphase microtubules:a comparative view.Current opinion in plant biology,6(6):568-576.
[20]Mineyuki,Y.(2007).Plant microtubule studies:past and present.Journal of plant research,120(1):45-51.
[21]Yuan,M.,Shaw,P.J.,Warn,R.M.,&Lloyd,C.W.(1994).Dynamic reorientation of cortical microtubules,from transverse to longitudinal,in living plant cells.Proceedings of the National Academy of Sciences,91(13):6,050-6,053.
[22]Bibikova,T.N.,Blancaflor,E.B.,&Gilroy,S.(1999).Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana.The Plant Journal,17(6):657-665.
[23]Whittington,A.T.,Vugrek,O.,Wei,K.J.,Hasenbein,N.G.,Sugimoto,K.,Rashbrooke,M.C.,&Wasteneys,G.O.(2001).MOR1 is essential for organizing cortical microtubules in plants.Nature,411(6837):610-613.
[24]Chan,J.,Mao,G.,Smertenko,A.,Hussey,P.J.,Naldrett,M.,Bottrill,A.,&Lloyd,C.W.(2003).Identification of a MAP65 isoform involved in directional expansion of plant cells.FEBS letters,534(1):161-163.
[25]Müller,S.,Smertenko,A.,Wagner,V.,Heinrich,M.,Hussey,P.J.,&Hauser,M.T.(2004).The plant microtubuleassociated protein At MAP65-3/PLE is essential for cytokinetic phragmoplast function.Current Biology,14(5):412-417.
[26]Wang,C.,Li,J.,&Yuan,M.(2007).Salt tolerance requires cortical microtubule reorganization in Arabidopsis.Plant and cell physiology,48(11):1,534-1,547.
[27]Caillaud,M.C.,Lecomte,P.,Jammes,F.,Quentin,M.,Pagnotta,S.,Andrio,E.,...&Favery,B.(2008).MAP65-3 microtubule-associated protein is essential for nematode-induced giant cell ontogenesis in Arabidopsis.The Plant Cell,20(2):423-437.
[28]Ji,S.,Lu,Y.,Li,J.,Wei,G.,Liang,X.,&Zhu,Y.(2002).Aβ-tubulin-like c DNA expressed specifically in elongating cotton fibers induces longitudinal growth of fission yeast.Biochemical and biophysical research communications,296(5):1,245-1,250.
[2]韩志国,张天真.棉纤维发育相关EST-SSR的特征功能及其定位[J].南京农业大学学报,2006,9(3):101-121.HAN Zhi-guo,ZHANG Tian-zhen.(2006).Cotton fiber development of EST-SSR function and localization features[J].Journal of Nanjing Agricultural University,9(3):101-121.(in Chinese)
[3]钱雯婕,王斐,李鸿彬.棉花单脱氢抗坏血酸还原酶基因的克隆及原核表达[J].西北农业学报,2012,21(5):118-122.QIAN Wen-jie,WANG Fei,LI Hong-bin.(2012).Gene clone and prokaryotic expression of Cotton single dehydroascorbate reductase[J].Acta Agriculturae Boreali-occidentalis Sinica,21(5):118-122.
[4]Smertenko,A.,Saleh,N.,Igarashi,H.,Mori,H.,HauserHahn,I.,Jiang,C.J.,...&Hussey,P.J.(2000).A new class of microtubule-associated proteins in plants.Nature cell biology,2(10):750-753.
[5]Ehrhardt,D.W.,&Shaw,S.L.(2006).Microtubule dynamics and organization in the plant cortical array.Annu.Rev.Plant Biol.,57:859-875.
[6]Nakajima,K.,Furutani,I.,Tachimoto,H.,Matsubara,H.,&Hashimoto,T.(2004).SPIRAL1 encodes a plant-specific microtubule-localized protein required for directional control of rapidly expanding Arabidopsis cells.The Plant Cell,16(5):1,178-1,190.
[7]Sedbrook,J.C.,Ehrhardt,D.W.,Fisher,S.E.,Scheible,W.R.,&Somerville,C.R.(2004).The Arabidopsis SKU6/SPIRAL1 gene encodes a plus end-localized microtubule-interacting protein involved in directional cell expansion.The Plant Cell,16(6):1,506-1,520.
[8]Nakajima,K.,Kawamura,T.,&Hashimoto,T.(2006).Role of the SPIRAL1 gene family in anisotropic growth of Arabidopsis thaliana.Plant and cell physiology,47(4):513-522.
[9]Bolton,J.J.,Soliman,K.M.,Wilkins,T.A.,&Jenkins,J.N.(2010).Aberrant Expression of Critical Genes during Secondary Cell Wall Biogenesis in a Cotton Mutant,Ligon Lintless-1.Comparative and functional genomics,2009.
[10]Shi,Y.H.,Zhu,S.W.,Mao,X.Z.,Feng,J.X.,Qin,Y.M.,Zhang,L.,...&Zhu,Y.X.(2006).Transcriptome profiling,molecular biological,and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.The Plant Cell,18(3):651-664.
[11]Li,X.B.,Cai,L.,Cheng,N.H.,&Liu,J.W.(2002).Molecular characterization of the cotton Gh TUB1 gene that is preferentially expressed in fiber.Plant Physiology,130(2):666-674.
[12]蒋建雄,张天真.利用CTAB/酸酚法提取棉花组织总RNA[J].棉花学报,2003,15(3):166-167.JIANG Jian-xiong,ZHANG Tian-zhen.(2003).Extraction of Total RNA in Cotton Tissues with CTAB-acidic Phenolic Method[J].Cotton Science,15(3):166-167.(in Chinese)
[13]田大鹏,葛娟石峰,等.棉花Gh DHAR2基因的克隆、功能序列分析及原核表达[J].生物技术通报,2012,(7):65-69.TIAN Da-peng,GE Juan,SHI Feng,et al.(2012).Cloning,Functional Sequence Analysis and Prokaryotic Expression of Cotton Gh DHAR2 c DNA[J].Biotechnology Bulletin,(7):65-69.(in Chinese)
[14]李学宁,杜军伟,李鸿彬.棉花脱氢抗坏血酸还原酶基因的克隆、原核表达与纯化[J].石河子大学学报(自然科学版),2010,28(5):542-545.LI Xue-ning,DU Jun-wei,LI Hong-bin.(2010).Cloning,Prokaryotic Expression and Purification of a Cotton Dehydroascorbate Reductase Gene[J].Journal of Shihezi University(Natural Science),28(5):542-545.(in Chinese)
[15]王芳,王斐,孙辉,等.棉花Gh DHAR3基因克隆、功能序列分析及烟草的遗传转化[J].西北农业学报,2011,20(5):88-93.WANG Fang,WANG Fei,SUN Hui,et al.(2011).Cloning and Sequence Analysis of a Cotton Ch DHAR3 c DNA and Genetic Transformation of Tobacco[J].Acta Agriculturae Boreali-occidentalis Sinica,20(5):88-93.(in Chinese)
[16]Cyr,R.J.,&Palevitz,B.A.(1995).Organization of cortical microtubules in plant cells.Current opinion in cell biology,7(1):65-71.
[17]Lloyd,C.(1994).Why should stationary plant cells have such dynamic microtubules.Molecular biology of the cell,5(12):1,277.
[18]Gundersen,G.G.,&Cook,T.A.(1999).Microtubules and signal transduction.Current opinion in cell biology,11(1):81-94.
[19]Hashimoto,T.(2003).Dynamics and regulation of plant interphase microtubules:a comparative view.Current opinion in plant biology,6(6):568-576.
[20]Mineyuki,Y.(2007).Plant microtubule studies:past and present.Journal of plant research,120(1):45-51.
[21]Yuan,M.,Shaw,P.J.,Warn,R.M.,&Lloyd,C.W.(1994).Dynamic reorientation of cortical microtubules,from transverse to longitudinal,in living plant cells.Proceedings of the National Academy of Sciences,91(13):6,050-6,053.
[22]Bibikova,T.N.,Blancaflor,E.B.,&Gilroy,S.(1999).Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana.The Plant Journal,17(6):657-665.
[23]Whittington,A.T.,Vugrek,O.,Wei,K.J.,Hasenbein,N.G.,Sugimoto,K.,Rashbrooke,M.C.,&Wasteneys,G.O.(2001).MOR1 is essential for organizing cortical microtubules in plants.Nature,411(6837):610-613.
[24]Chan,J.,Mao,G.,Smertenko,A.,Hussey,P.J.,Naldrett,M.,Bottrill,A.,&Lloyd,C.W.(2003).Identification of a MAP65 isoform involved in directional expansion of plant cells.FEBS letters,534(1):161-163.
[25]Müller,S.,Smertenko,A.,Wagner,V.,Heinrich,M.,Hussey,P.J.,&Hauser,M.T.(2004).The plant microtubuleassociated protein At MAP65-3/PLE is essential for cytokinetic phragmoplast function.Current Biology,14(5):412-417.
[26]Wang,C.,Li,J.,&Yuan,M.(2007).Salt tolerance requires cortical microtubule reorganization in Arabidopsis.Plant and cell physiology,48(11):1,534-1,547.
[27]Caillaud,M.C.,Lecomte,P.,Jammes,F.,Quentin,M.,Pagnotta,S.,Andrio,E.,...&Favery,B.(2008).MAP65-3 microtubule-associated protein is essential for nematode-induced giant cell ontogenesis in Arabidopsis.The Plant Cell,20(2):423-437.
[28]Ji,S.,Lu,Y.,Li,J.,Wei,G.,Liang,X.,&Zhu,Y.(2002).Aβ-tubulin-like c DNA expressed specifically in elongating cotton fibers induces longitudinal growth of fission yeast.Biochemical and biophysical research communications,296(5):1,245-1,250.