铁棍山药SUS基因CDS区克隆与生物信息学分析
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摘要
目的:对铁棍山药蔗糖合成酶(sucrose synthase,SUS)基因的编码区域(coding sequence,CDS)进行克隆和蛋白质结构分析,为该基因的调控机制与山药多糖的合成机制提供理论依据。方法:提取铁棍山药总RNA并反转录为cDNA第一链,根据本实验室铁棍山药基因组数据经注释得到的SUS基因序列设计一对特异性引物,利用基因克隆技术获得SUS基因的编码区域并通过蛋白质预测分析软件分析蛋白质序列特征。结果:克隆得到一个长度2 448 bp的基因序列,具有一个完整的开放阅读框架(open reading frame,ORF),命名为DoSUS1。DoSUS1的分子式为C_(4209)H_(6534)N_(1115)O_(1205)S_(23),相对分子质量9 788.32,共815个氨基酸,理论等电点(PI)6.10,消光系数为110 505,脂溶性指数(AI)为94.15,不稳定指数为32.18,平均亲水指数(GRAVY)为-0.225,属于稳定可溶性酸性蛋白质。DoSUS1氨基酸序列存在多个磷酸化位点,不存在跨膜区与信号肽。蛋白质二级结构与三级结构结果显示DoSUS1属于全α类蛋白质。功能域预测结果显示DoSUS1有蔗糖合成与糖基转移两个功能域。同源性比对结果显示DoSUS1的氨基酸序列与所比对单子叶植物的氨基酸序列相似性均>80%。系统进化树显示DoSUS1与单子叶植物的SUS进化关系较近。结论:首次从铁棍山药中克隆出SUS基因的编码序列,并对其蛋白质结构进行了分析,为进一步阐明SUS在山药生长发育和多糖合成机制中的作用奠定了基础。
Objective: To clone CDS sequence of Dioscorea opposita SUS gene and analyze the protein structure,in order to provide a theoretical basis for the regulation mechanism of SUS gene and the synthesis mechanism of D. opposita polysaccharides. Method: Total RNA in D. opposita was extracted and reversetranscribed into first strand of cDNA. Specific primers were designed according to an annotated SUS gene sequence obtained from the laboratory D. opposita genome database,and the coding region of the SUS gene was obtained by gene cloning technique and the protein sequence characteristics were analyzed by protein prediction analysis software. Result: A 2 448 bp gene sequence was cloned with a complete open reading frame( ORF). The gene was named DoSUS1. The formula of protein encoded by DoSUS1 gene in D. opposita was C_(4209)H_(6534)N_(1115)O_(1205)S_(23),and the molecular weight was 9 788. 32,the total number of amino acids was 815,the theory isoelectric point( PI) was 6. 10,the extinction coefficient was 110 505,the aliphatic index( AI) was 94. 15,the instability index was 32. 18,and the grand average of hydropathicity( GRAVY) was-0. 225. It was a stable and soluble acidic protein. There were phosphorylation sites in the DoSUS1 amino acid sequence, with no transmembrane region and signal peptide. The secondary structure and the tertiary structure showed that DoSUS1 was an α class protein. Functional domain predictions showed that DoSUS1 had sucrose synthesis domain and glycosyl transfer domain. The homology alignment showed that the amino acid sequence of DoSUS1 was more than 80% similar to the amino acid sequence of the aligned monocots. The phylogenetic tree showed that DoSUS1 was closely related to SUS of monocotyledon evolution. Conclusion: The coding sequence of SUS gene was cloned from D. opposita for the first time,and its protein structure was analyzed to lay a foundation for further studying the roles of SUS in the growth and polysaccharide synthesis of D. opposita.
Objective: To clone CDS sequence of Dioscorea opposita SUS gene and analyze the protein structure,in order to provide a theoretical basis for the regulation mechanism of SUS gene and the synthesis mechanism of D. opposita polysaccharides. Method: Total RNA in D. opposita was extracted and reversetranscribed into first strand of cDNA. Specific primers were designed according to an annotated SUS gene sequence obtained from the laboratory D. opposita genome database,and the coding region of the SUS gene was obtained by gene cloning technique and the protein sequence characteristics were analyzed by protein prediction analysis software. Result: A 2 448 bp gene sequence was cloned with a complete open reading frame( ORF). The gene was named DoSUS1. The formula of protein encoded by DoSUS1 gene in D. opposita was C_(4209)H_(6534)N_(1115)O_(1205)S_(23),and the molecular weight was 9 788. 32,the total number of amino acids was 815,the theory isoelectric point( PI) was 6. 10,the extinction coefficient was 110 505,the aliphatic index( AI) was 94. 15,the instability index was 32. 18,and the grand average of hydropathicity( GRAVY) was-0. 225. It was a stable and soluble acidic protein. There were phosphorylation sites in the DoSUS1 amino acid sequence, with no transmembrane region and signal peptide. The secondary structure and the tertiary structure showed that DoSUS1 was an α class protein. Functional domain predictions showed that DoSUS1 had sucrose synthesis domain and glycosyl transfer domain. The homology alignment showed that the amino acid sequence of DoSUS1 was more than 80% similar to the amino acid sequence of the aligned monocots. The phylogenetic tree showed that DoSUS1 was closely related to SUS of monocotyledon evolution. Conclusion: The coding sequence of SUS gene was cloned from D. opposita for the first time,and its protein structure was analyzed to lay a foundation for further studying the roles of SUS in the growth and polysaccharide synthesis of D. opposita.
引文
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[3]ZHOU L,Paull R E.Sucrose metabolism during Papaya(Corica papaya)fruit growth and ripening[J].J Am Soc Hort Sci,2001,126(3):351-357.
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[12]Afoakwa E O,Sefa-Dedeh S.Chemical composition and quality changes occurring in Dioscorea dumetorum pax tubers after harvest[J].Food Chem,2001,75(1):85-91.
[13]ZHANG Z S,WANG X M,LIU C B,et al.The degradation,antioxidant and antimutagenic activity of the mucilage polysaccharide from Dioscorea opposita[J].Carbohyd Polym,2016,150(20):227-231.
[14]LIU Y X,LI H F,FAN Y Y,et al.Antioxidant and antitumor activities of the extracts from Chinese Yam(Dioscorea opposite Thunb.)flesh and peel and the effective compounds[J].Food Sci,2016,81(6):1553-1564.
[15]NIU X,HE Z,LI W,et al.Immunomodulatory activity of the glycoprotein isolated from the Chinese Yam(Dioscorea opposita Thunb)[J].Phytother Res,2017,31(10):1557-1563.
[16]Vauglm M W,Harrington G N,Bush D R.Sucrosemediated transcription alregulation of sucrose symporter activity in the Phloem[J].Proc Natl Acad Sci USA,2002,99(16):10876-10870.
[17]周生茂.山药不同基因型地下块茎糖类和酚类物质形成、调控及相关基因分离的研究[D].杭州:浙江大学,2009.
[18]Rost B,Sander C.Combining evolutionary information and neural networks to predict protein secondary structure[J].Protein Sci,1994,19(1):55-72.
[19]Schm9lzer K,Gutmann A,Diricks M,et al.Sucrose synthase:A unique glycosyltransferase for biocatalytic glycosylation process development[J].Biotechnol Adv,2016,34(2):88-111.
[20]HU L,LIU S.Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers[J].Genet Mol Biol,2011,34(4):624-634.
[21]Hardin S C,Winter H,Huber S C.Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity[J].Plant Physiol,2004,134(4):1427-1438.
[22]Anguenot R,Nguyen-Quoc B,Yelle S,et al.Protein phosphory lationand membrane association ofsucrose synthasein developing tomato fruit[J].Plant Physiol Bioch,2006,44(5/6):294-300.
[23]严亮.中国传统兰科药用植物铁皮石斛基因组及其生物学特性研究[D].长春:吉林大学,2014.
[24]王凌云,郭明,赵艳,等.谷子蔗糖合成酶基因家族鉴定及生物信息学分析[J].江苏农业科学,2017,45(15):30-34.
[25]LI C R,ZHANG X B,HUANG C H,et al.Cloning,characterization and tissue Specific expression of a sucrose synthase gene from tropical epiphytic CAM Orchid Mokara yellow[J].J Plant Physiol,2004,161(1):87-94.
[26]Verma A K,Upadhyay S K,Verma P C,et al.Functional analysis of sucrose phosphate synthase(SPS)and sucrose synthase(SUS)in sugarcane(Saccharum)cultivars[J].Plant Biol,2011,13(2):325-332.
[27]张秀梅,杜丽清,孙光明,等.菠萝果实发育过程中糖积累与其代谢酶的关系[J].热带作物学报,2008,29(1):10-13.
[2]Elling L.Effect of metal ions on suctose synthase from rice grains-a gtudy on enzyme inhibition and enzyme topography[J].Glycobiology,1995,5(2):20l-206.
[3]ZHOU L,Paull R E.Sucrose metabolism during Papaya(Corica papaya)fruit growth and ripening[J].J Am Soc Hort Sci,2001,126(3):351-357.
[4]Geigenberger P,Stitt M.Sucrose synthase catalyses a readily reversible reaction in developing potato tuber and other plant tissues[J].Planta,1993,189(3):329-339.
[5]RUAN Y L,Churey P S.Afiberless seed mutation in cotton is associated with lack of fiberinitiation in ovulee pidermis and alterations in sucrose synthaseexpression and carbon partitioningin developing seeds[J].Plant Physiol,1998,118(2):399-406.
[6]Jha A B,Dubey R S.Carbohydrate metabolism in growing rice seedlings under arsenic toxicity[J].J Plant Physiol,2004,161(7):886-872.
[7]Slugina M A,Boris K V,Kakimzhanova A A,et al.Intraspecific polymorphism of the sucrose synthase genes in Russian and Kazakhstan potato cultivars[J].Genetika,2014,50(6):677-682.
[8]JIANG Q,HOU J,HAO C,et al.The wheat(T.aestivum)sucrose synthase 2 gene(Ta SUS2)active in endosperm development is associated with yield traits[J].Funct Integr Genomics,2010,11(1):49-61.
[9]Horst I,Welham T,Kelly S,et al.TILLING mutants of Lotus japonicus reveal that nitrogen assimilation and fixation can occur in the absence of nodule-enhanced sucrose synthase[J].Plant Physiol,2007,144(2):806-820.
[10]Iwona C,Leszek A,Kleczkowski.Expression of several genes involved in sucrose/starch metabolism as affected by different strategies to induce phosphate deficiency in Arabidopsis[J].Acta Physiol Plant,2005,27(20):147-155.
[11]王海平,沈镝.山药种质资源描述规范和数据标准[M].北京:中国农业科学技术出版社,2014:1-6.
[12]Afoakwa E O,Sefa-Dedeh S.Chemical composition and quality changes occurring in Dioscorea dumetorum pax tubers after harvest[J].Food Chem,2001,75(1):85-91.
[13]ZHANG Z S,WANG X M,LIU C B,et al.The degradation,antioxidant and antimutagenic activity of the mucilage polysaccharide from Dioscorea opposita[J].Carbohyd Polym,2016,150(20):227-231.
[14]LIU Y X,LI H F,FAN Y Y,et al.Antioxidant and antitumor activities of the extracts from Chinese Yam(Dioscorea opposite Thunb.)flesh and peel and the effective compounds[J].Food Sci,2016,81(6):1553-1564.
[15]NIU X,HE Z,LI W,et al.Immunomodulatory activity of the glycoprotein isolated from the Chinese Yam(Dioscorea opposita Thunb)[J].Phytother Res,2017,31(10):1557-1563.
[16]Vauglm M W,Harrington G N,Bush D R.Sucrosemediated transcription alregulation of sucrose symporter activity in the Phloem[J].Proc Natl Acad Sci USA,2002,99(16):10876-10870.
[17]周生茂.山药不同基因型地下块茎糖类和酚类物质形成、调控及相关基因分离的研究[D].杭州:浙江大学,2009.
[18]Rost B,Sander C.Combining evolutionary information and neural networks to predict protein secondary structure[J].Protein Sci,1994,19(1):55-72.
[19]Schm9lzer K,Gutmann A,Diricks M,et al.Sucrose synthase:A unique glycosyltransferase for biocatalytic glycosylation process development[J].Biotechnol Adv,2016,34(2):88-111.
[20]HU L,LIU S.Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers[J].Genet Mol Biol,2011,34(4):624-634.
[21]Hardin S C,Winter H,Huber S C.Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity[J].Plant Physiol,2004,134(4):1427-1438.
[22]Anguenot R,Nguyen-Quoc B,Yelle S,et al.Protein phosphory lationand membrane association ofsucrose synthasein developing tomato fruit[J].Plant Physiol Bioch,2006,44(5/6):294-300.
[23]严亮.中国传统兰科药用植物铁皮石斛基因组及其生物学特性研究[D].长春:吉林大学,2014.
[24]王凌云,郭明,赵艳,等.谷子蔗糖合成酶基因家族鉴定及生物信息学分析[J].江苏农业科学,2017,45(15):30-34.
[25]LI C R,ZHANG X B,HUANG C H,et al.Cloning,characterization and tissue Specific expression of a sucrose synthase gene from tropical epiphytic CAM Orchid Mokara yellow[J].J Plant Physiol,2004,161(1):87-94.
[26]Verma A K,Upadhyay S K,Verma P C,et al.Functional analysis of sucrose phosphate synthase(SPS)and sucrose synthase(SUS)in sugarcane(Saccharum)cultivars[J].Plant Biol,2011,13(2):325-332.
[27]张秀梅,杜丽清,孙光明,等.菠萝果实发育过程中糖积累与其代谢酶的关系[J].热带作物学报,2008,29(1):10-13.