霍山石斛GDP-甘露糖焦磷酸化酶基因克隆及表达分析
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摘要
为明确GDP-甘露糖焦磷酸化酶(GMPP)基因的功能及其在石斛多糖生物合成中的表达调控机制,该研究以霍山石斛为原材料,克隆GMPP基因且对该c DNA序列进行分析,利用qPCR技术检测霍山产地3种石斛(霍山石斛、铁皮石斛、铜皮石斛)中GMPP基因的表达情况。结果显示霍山石斛GMPP基因c DNA序列长1 867 bp,包含1 245 bp的开放阅读框(ORF),编码415个氨基酸;序列分析表明霍山石斛GMPP与铁皮石斛、黄花石斛相似性高达99%;进化树分析该基因与铁皮石斛、黄花石斛等植物的GMPP亲缘关系较近; qPCR结果显示GMPP基因在霍山石斛茎中表达量最高,其次是叶,然后是花,根中表达量最低,且在霍山产3种石斛中霍山石斛的相对表达量最高,铁皮石斛的表达量最低。该研究发现霍山石斛GMPP基因表达水平与其茎、叶、花、根中多糖含量呈高度相关性,对进一步研究霍山石斛多糖生物合成机制将起到积极的促进作用。
In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.
In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.
引文
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[22]张炜玲,王新生,戴亚峰,等.响应面法优化霍山石斛多糖提取工艺[J].安徽农业学,2012,40(12):7061.
[23]黄静,李胜立,赵宏伟,等.霍山石斛多糖对四氯化碳致急性肝损伤小鼠的保护作用[J].中国中药杂志,2013,38(4):528.
[24]王超群,李德文,袁晨琳,等.霍山石斛多糖不同组分理化性质及免疫活性的比较研究[J].安徽农业科学,2018,46(13):160.
[25]徐海军,高温婷,杨健,等.霍山石斛多糖对衰老模型大鼠肠道消化吸收功能和形态结构的影响[J].皖西学院学报,2018,34(2):1.
[26] Li F,Cui S H,Zha X Q,et al. Structure and bioactivity of a polysaccharide extracted from protocorm-like bodies of Dendrobium huoshanense[J]. Int J Biol Macromol,2015,72(6):664.
[27] Si H Y,Chen N F,Chen N D,et al. Structural characterisation of a water-soluble polysaccharide from tissue-cultured Dendrobium huoshanense C. Z. Tang et S. J. Cheng[J]. Nat Prod Res,2017,32(3):252.
[28]邓辉,韩邦兴,谷仿丽,等.安徽霍山产3种石斛多糖组成的比较研究[J].中药材,2017,40(6):1300.
[29] Fey S,Elling L,Kragl U. The cofactor Mg2+--a key switch for effective continuous enzymatic production of GDP-mannose using recombinant GDP-mannose pyrophosphorylase[J]. Carbohyd Res,1997,305(3):475.
[30] Asención Diez M D,Demonte A,Giacomelli J,et al. Functional characterization of GDP-mannose pyrophosphorylase from leptospira interrogans serovar copenhageni[J]. Arch Microbiol,2010,192(2):103.
[31] Reyes F,Orellana A. Golgi transporters:opening the gate to cell wall polysaccharide biosynthesis[J]. Curr Opin Plant Biol,2008,11(3):244.
[32] Keller R,Renz F S,Kossmann J. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence[J]. Plant J,1999,19(2):131.
[33]王维鹏,郭新波,唐克轩.拟南芥GDP-甘露糖焦磷酸化酶基因转化生菜的研究[J].上海交通大学学报:农业科学版,2011,29(2):43.
[34]周桂芬,庞敏霞,陈素红,等.铁皮石斛茎、叶多糖含量及多糖部位柱前衍生化-高效液相色谱指纹图谱比较研究[J].中国中药杂志,2014,39(5):795.
[35]李文静,李进进,李桂锋,等.GC-MS分析4种石斛花挥发性成分[J].中药材,2015,38(4):777.
[36]陈乃东,李俊.霍山石斛野生植株·组培植株与河南石斛叶表皮微形态特征研究[J].安徽农业科学,2016,44(25):186.
[37]王楠楠,戴明珠,徐俞悦,等.铁皮石斛花对肾上腺皮质激素致肾阴虚模型小鼠的影响[J].中药药理与临床,2017,33(1):116.
[38]梁凯伦,方萍,施秋秋,等.铁皮石斛花对高糖高脂饮酒致高血压大鼠的降压作用及机制研究[J].中国中药杂志,2018,43(1):147.
[2]童晨曦,齐蕾.霍山石斛的研究进展及可持续发展[J].医学综述,2015,21(24):4512.
[3]崔胜文,罗双群,周婧琪,等.霍山石斛多糖研究进展[J].食品研究与开发,2018,39(8):207.
[4]陈存武,戴军,姚厚军,等.霍山石斛资源与鉴定研究进展[J].现代中药研究与实践,2013,27(3):84.
[5]徐光涛.提高霍山石斛仿生态栽培成活率方法的研究[J].安徽林业科技,2011,37(6):62.
[6]钱文林,张建霞,吴坤林,等.霍山石斛种苗繁殖与栽培研究[J].热带亚热带植物学报,2013,21(3):240.
[7] Xing X H,Cui S W,Nie S,et al. A review of isolation process,structural characteristics,and bioactivities of water-soluble polysaccharides from Dendrobium plants[J]. Bioactive Carbohy drates and Dietary Fibre,2013,2(1):131.
[8] Barth C,Gouzd Z A,Steele H P,et al. A mutation in GDPmannose pyrophosphorylase causes conditional hypersensitivity to ammonium,resulting in Arabidopsis root growth inhibition,altered ammonium metabolism,and hormone homeostasis[J].J Exp Bot,2010,61(2):379.
[9]王维鹏,郭新波,唐克轩.拟南芥GDP-甘露糖焦磷酸化酶基因转化生菜的研究[J].上海交通大学学报,2011,29(2):43.
[10]肖瑶,周天山,李佼,等.茶树GDP-D-甘露糖焦磷酸化酶基因c DNA全长的克隆与表达分析[J].茶叶科学,2015,35(1):55.
[11]吴海琴,赵瑛,符庆功,等.番茄叶片GDP-甘露糖焦磷酸化酶基因表达载体的构建与转化[J].生物技术通报,2010(2):154.
[12]申艳红,陈晓静,卢秉国,等.番木瓜GDP-D-甘露糖焦磷酸化酶基因的克隆及分析[J].福建农林大学学报,2011,40(2):172.
[13] He C,Yu Z,Teixeira da Silva J A,et al. Do GMP1 from Dendrobium officinale contributes to mannose content of water-soluble polysaccharides and plays a role in salt stress response[J]. Sci Rep,2017,7(10):1.
[14] Liu L,Han R,Yu N,et al. A method for extracting high-quality total RNA from plant rich in polysaccharides and polyphenols using Dendrobium huoshanense[J]. PLoS ONE,2018,13(5):1.
[15] Han R,Xie D,Tong X,et al. Transcriptomic landscape of Dendrobium huoshanense and its genes related to polysaccharide biosynthesis[J]. Acta Soc Bot Pol,2018,87(1):1.
[16] He C,Zhang J,Liu X,et al. Identification of genes involved in biosynthesis of mannan polysaccharides in Dendrobium officinale by RNA-seq analysis[J]. Plant Mol Biol,2015,88(3):219.
[17] Buenavista M T,Roche D B. Improvement of 3D protein models using multiple templates guided by single-template model quality assessment[J]. Bioinformatics(Oxford,England),2012,28(14):1851.
[18] Mc Guffin L J,Atkins J D,Salehe B R,et al. IntFOLD:an integrated server for modelling protein structures and functions from amino acid sequences[J]. Nucleic Acids Res,2015,43(W1):169.
[19] Mc Guffin L J,Shuid A N,Kempster R,et al. Accurate template-based modeling in CASP12 using the IntFOLD4-TS,ModFOLD6,and Re FOLD methods[J]. Proteins,2018,86(S1):335.
[20]金海英,张炜玲,戴亚锋,等.霍山产3种石斛多糖含量变化规律[J].安徽农业科学,2012,40(9):5152.
[21]包雪声,顺庆生,陈立钻.中国药用石斛[M].上海:复旦大学出版社,2001:75.
[22]张炜玲,王新生,戴亚峰,等.响应面法优化霍山石斛多糖提取工艺[J].安徽农业学,2012,40(12):7061.
[23]黄静,李胜立,赵宏伟,等.霍山石斛多糖对四氯化碳致急性肝损伤小鼠的保护作用[J].中国中药杂志,2013,38(4):528.
[24]王超群,李德文,袁晨琳,等.霍山石斛多糖不同组分理化性质及免疫活性的比较研究[J].安徽农业科学,2018,46(13):160.
[25]徐海军,高温婷,杨健,等.霍山石斛多糖对衰老模型大鼠肠道消化吸收功能和形态结构的影响[J].皖西学院学报,2018,34(2):1.
[26] Li F,Cui S H,Zha X Q,et al. Structure and bioactivity of a polysaccharide extracted from protocorm-like bodies of Dendrobium huoshanense[J]. Int J Biol Macromol,2015,72(6):664.
[27] Si H Y,Chen N F,Chen N D,et al. Structural characterisation of a water-soluble polysaccharide from tissue-cultured Dendrobium huoshanense C. Z. Tang et S. J. Cheng[J]. Nat Prod Res,2017,32(3):252.
[28]邓辉,韩邦兴,谷仿丽,等.安徽霍山产3种石斛多糖组成的比较研究[J].中药材,2017,40(6):1300.
[29] Fey S,Elling L,Kragl U. The cofactor Mg2+--a key switch for effective continuous enzymatic production of GDP-mannose using recombinant GDP-mannose pyrophosphorylase[J]. Carbohyd Res,1997,305(3):475.
[30] Asención Diez M D,Demonte A,Giacomelli J,et al. Functional characterization of GDP-mannose pyrophosphorylase from leptospira interrogans serovar copenhageni[J]. Arch Microbiol,2010,192(2):103.
[31] Reyes F,Orellana A. Golgi transporters:opening the gate to cell wall polysaccharide biosynthesis[J]. Curr Opin Plant Biol,2008,11(3):244.
[32] Keller R,Renz F S,Kossmann J. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence[J]. Plant J,1999,19(2):131.
[33]王维鹏,郭新波,唐克轩.拟南芥GDP-甘露糖焦磷酸化酶基因转化生菜的研究[J].上海交通大学学报:农业科学版,2011,29(2):43.
[34]周桂芬,庞敏霞,陈素红,等.铁皮石斛茎、叶多糖含量及多糖部位柱前衍生化-高效液相色谱指纹图谱比较研究[J].中国中药杂志,2014,39(5):795.
[35]李文静,李进进,李桂锋,等.GC-MS分析4种石斛花挥发性成分[J].中药材,2015,38(4):777.
[36]陈乃东,李俊.霍山石斛野生植株·组培植株与河南石斛叶表皮微形态特征研究[J].安徽农业科学,2016,44(25):186.
[37]王楠楠,戴明珠,徐俞悦,等.铁皮石斛花对肾上腺皮质激素致肾阴虚模型小鼠的影响[J].中药药理与临床,2017,33(1):116.
[38]梁凯伦,方萍,施秋秋,等.铁皮石斛花对高糖高脂饮酒致高血压大鼠的降压作用及机制研究[J].中国中药杂志,2018,43(1):147.