植物生理生化与分子生物学

砀山酥梨多酚氧化酶基因(PbPPO)的原核表达及优化研究

  • 常 艳 ,
  • 刘晓峰 ,
  • 金 青 ,
  • 蔡永萍 ,
  • 聂 凡 ,
  • 林 毅
展开
  • (1.安徽农业大学生命科学学院,安徽 合肥230036;2.安徽省农业科学院园艺研究所,安徽 合肥 230031)
常艳,硕士,实验师,从事植物分子生物学研究。

收稿日期: 2017-06-28

  修回日期: 2017-08-02

  网络出版日期: 2017-09-30

基金资助

国家自然科学基金(No. 31640068)

Prokaryotic Expression and Its Optimization of Polyphenol Oxidase Gene (PbPPO) in Pyrus bretschneideri ‘Dangshan Su’

  • CHANG Yan ,
  • LIU Xiao-Feng ,
  • JIN Qing ,
  • CAI Yong-Ping ,
  • NIE Fan ,
  • LIN Yi
Expand
  • (1.School of Life Sciences, Anhui Agricultural University, Hefei 230036, Anhui China; 2.Horticultural Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui China)

Received date: 2017-06-28

  Revised date: 2017-08-02

  Online published: 2017-09-30

摘要

从砀山酥梨Pyrus bretschneideri ‘Dangshan Su’果实中克隆得到一条多酚氧化酶基因(PbPPO)的全长cDNA序列(GenBank登录号:JF809859)。通过生物信息学分析表明,PbPPO基因CDS区全长1782 bp,编码593个氨基酸,氨基酸序列由N端叶绿体转运肽、Cu结合区与C端疏水区三部分组成。为进一步研究PbPPO基因的功能,成功构建其原核表达载体pET-28a-PbPPO,并在大肠杆菌Rosetta菌株中成功诱导表达,经优化后显示,在28 ℃、1.0 mmol·L-1 IPTG诱导5 h的条件下,融合蛋白表达量最高。

本文引用格式

常 艳 , 刘晓峰 , 金 青 , 蔡永萍 , 聂 凡 , 林 毅 . 砀山酥梨多酚氧化酶基因(PbPPO)的原核表达及优化研究[J]. 亚热带植物科学, 2017 , 46(03) : 209 -214 . DOI: 10.3969/j.issn.1009-7791.2017.03.002

Abstract

This study cloned the full-length cDNA sequence of a polyphenol oxidase gene (PbPPO) from Dangshan Su pear. The bioinformatics analysis showed that the full-length of PbPPO gene was 1782 bp, encoding 593 amino acids. The amino acid sequence consisted of three parts: N terminal chloroplast transport peptide, Cu binding region and C-terminal hydrophobic region. In order to further study the function of PbPPO gene, the prokaryotic expression vector pET-28a-PbPPO was successfully constructed, and the fusion protein was successfully expressed in Escherichia coli Rosetta. After optimization, it was proved that the fusion protein had the highest expression at 28 ℃ and 1.0 mmol·L-1 IPTG for 5 h. This study not only created the conditions for the study of PbPPO enzymatic properties, but also provided the scientific basis for the molecular regulation of stone cell content and the improvement of Dangshan Su pear quality.

参考文献

[1] 程俊,程曦,盛玲玲,方志,张金云,金青,蔡永萍,林毅. 砀山酥梨肉桂酸4-羟化酶基因的克隆及表达分析[J]. 农业生物技术学报, 2016,24(11): 1698—1708.
[2] 田路明,曹玉芬,高源,董星光. 梨品种果肉石细胞团大小对果肉质地的影响[J]. 园艺学报, 2011,38(7): 1225—1234.
[3] Cheng X, Yan C C, Zhang J Y, Ma C H, Li S M, Jin Q, Zhang N, Cao Y P, Lin Y, Cai Y P. The effect of different pollination on the expression of Dangshan Su pear microRNA[J]. BioMed Research International, 2017,2017(2): 1—18.
[4] Cai Y P, Li G Q, Nie J Q, Lin Y, Nie F. Study of the structure and biosynthetic pathway of lignin in stone cells of pear[J]. Scientia Horticulturae, 2010,125(3): 374—379.
[5] Tao S, Shahrokh K, Zhang H, Zhang S. Anatomy, ultrastructure and lignin distribution of stone cells in two Pyrus species[J]. Plant Science, 2009,176(3): 413—419.
[6] Yan C C, Yin M, Zhang N, Jin Q, Fang Z, Lin Y, Cai Y P. Stone cell distribution and lignin structure in various pear varieties[J]. Scientia Horticulturae, 2014,174(1): 142—150.
[7] Barros J, Serk H, Granlund I, Pesquet E. The cell biology of lignification in higher plants[J]. Annals of Botany, 2015,115(7): 1053—1074.
[8] Ren G, Wang B, Zhu X, Mu Q, Wang C, Tao R, Fang J G. Cloning, expression, and characterization of miR058 and its target PPO, during the development of grapevine berry stone[J]. Gene, 2014,548(2): 166—173.
[9] 陈晓亚. 植物次生代谢及调控[M]. 北京: 科学出版杜, 1998: 390—401.
[10] Campbell M M, Sederoff R R. Variation in lignin content and composition[J]. Plant Physiology, 1996,110(1): 3—13.
[11] 贺立红,宾金华. 高等植物中的多酚氧化酶[J]. 植物生理学报, 2001,37(4): 340—345.
[12] Hunt M D, Eannetta N T, Yu H, Newman S M, Steffens J C. cDNA cloning and expression of potato polyphenol oxidase[J]. Plant Molecular Biology, 1993,21(1): 59—68.
[13] Dry I B, Robinson S P. Molecular cloning and characterisation of grape berry polyphenol oxidase[J]. Plant Molecular Biology, 1994,26(1): 495—502.
[14] 刘国强,吴锦程,唐朝晖,张琳华,戴巧斌. 枇杷采后酚类物质代谢与果肉木质化的关系[J]. 中国农学通报, 2008,24(1): 247—251.
[15] 芮怀瑾,汪开拓,尚海涛,唐双双,金鹏,曹士峰,郑永华. 热处理对冷藏枇杷木质化及相关酶活性的影响[J]. 农业工程学报, 2009,25(7): 294—298.
[16] Bachem C W B, Speckmann G J, Linde P C G V D, Verheggen F T M, Hunt M D, Steffens J C, Marc Z J C. Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in potato tubers[J]. Nature Biotechnology, 1994,12(11): 1101—1105.
[17] 王清,黄惠英,马文芳. 反义PPO基因对马铃薯块茎褐化的影响[J]. 作物学报, 2007,33(11): 1822—1827.
[18] Masatsune M, Miyoshi H, Nanae M. Transgenic apple (Malus × domestica) shoot showing low browning potential[J]. Journal of Agricultural & Food Chemistry, 2000,48(11): 5243—5248.
[19] Li L, Steffens J C. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance[J]. Planta, 2002,215(2): 239—247.
[20] Wang J, Constabel C P. Polyphenol oxidase overexpression in transgenic Populus enhances resistance to herbivory by forest tent caterpillar (Malacosoma disstria)[J]. Planta, 2004,220(1): 87—96.
[21] Chevalier T, De R D, Mbéguiéambéguié D, Gauillard F, Richard Forget F, Fils Lycaon B R. Molecular cloning and characterization of apricot fruit polyphenol oxidase[J]. Plant Physiology, 1999,119(4): 1261—1269.
[22] Kim J Y, Seo Y S, Kim J E, Kim J E, Sung S K, Song K J, An G, Kim W T. Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple[J]. Plant Science, 2001,161(6): 1145—1152.
[23] Haruta M, Murata M, Kadokura H, Homma S. Immunological and molecular comparison of polyphenol oxidase in rosaceae fruit trees[J]. Phytochemistry, 1999,50(6): 1021—1025.
[24] Nakayama T, Yonekura-Sakakibara K, Sato T, Kikuchi S, Fukui Y, Fukuchi-Mizutani M, Ueda T, Nakao M, Tanaka Y, Kusumi T, Nishino T. Aureusidin synthase: A polyphenol oxidase homolog responsible for flower coloration[J]. Science, 2000,290(5494): 1163—1169.
[25] 陈东生,王坤波,黄建安,刘仲华. 茶树多酚氧化酶研究进展[J]. 茶叶通讯, 2012,39(2): 17—21.
[26] Pedersen S. Escherichia coli ribosomes translate in vivo with variable rate[J]. Embo Journal, 1984,3(12): 2895—2898.
[27] Pohlner J, Meyer T F, Jalajakumari M B, Manning P A. Nucleotide sequence of ompV, the gene for a major vibrio cholerae outer membrane protein[J]. Molecular & General Genetics, 1986,205(3): 494—500.
[28] S?rensen M A, Kurland C G, Pedersen S. Codon usage determines translation rate in Escherichia coli[J]. Journal of Molecular Biology, 1989,207(2): 365—377.
[29] 陈娇,袁德保,谭琳,杨昭,李奕星,王朝政,李芬芳,仇厚援,陈文学. 香蕉PPO基因的克隆及原核表达[J]. 贵州农业科学, 2015,43(7): 14—18.
[30] 王乃栋,张丽霞,向勤锃,李巍巍. 茶树多酚氧化酶基因的生物信息学分析及原核表达[J]. 茶叶科学, 2011,31(1): 33—39.
文章导航

/