食品科学 ›› 2019, Vol. 40 ›› Issue (6): 203-208.doi: 10.7506/spkx1002-6630-20180125-328

• 生物工程 • 上一篇    下一篇

苹果根皮苷-2-O-糖基转移酶基因克隆与表达模式分析

冉军舰1,梁新红1,陈晓静2,李向阳3,焦凌霞1,赵瑞香1,*   

  1. 1.河南科技学院食品学院,河南省高校重点实验室培育基地,新乡市农产品加工工程技术研究中心,河南 新乡 453003;2.河南科技学院新科学院,河南 新乡 453003;3.新乡职业技术学院后勤管理处,河南 新乡 453003
  • 出版日期:2019-03-25 发布日期:2019-04-02
  • 基金资助:
    河南省高校重点科研项目(18A550006);河南省高校科技创新人才支持计划项目(17HASTIT037);河南科技学院高层次人才科研项目(2015016)

Cloning and Expression Profile Analysis of the Phloridzin 2’-O-Glycosyltransferase Gene in Apple

RAN Junjian1, LIANG Xinhong1, CHEN Xiaojing2, LI Xiangyang3, JIAO Lingxia1, ZHAO Ruixiang1,*   

  1. 1. Key Lab Breeding Base of College of Henan Province, Xinxiang Engineering Technology Research Center for Agricultural Products Processing, School of Food Science, Henan Institute of Science and Technology, Xinxiang 453003, China; 2. Xinke College, Henan Institute of Science and Technology, Xinxiang 453003, China; 3. Rear Service Management, Xinxiang Vocational and Technical College, Xinxiang 453003, China
  • Online:2019-03-25 Published:2019-04-02

摘要: 目的:根皮苷-2-O-糖基转移酶(phloridzin 2’-O-glycosyltransferase,P2’-GT)是根皮苷合成最后一步关键酶,可以把根皮素转化成根皮苷,本研究在富士苹果中克隆出P2’-GT基因,对基因编码产物进行生物信息学分析和基因的表达模式分析。方法:以苹果皮为材料,提取RNA反转录合成cDNA为模板,设计特异性引物进行扩增和测序,利用Pro Param tool、TMHMM等在线软件对编码蛋白进行生物信息学分析,利用实时荧光定量聚合酶链式反应(realtime fluorescence quatitative polymerase chain reaction,RTFQ-PCR)分析P2’-GT在苹果不同部位、不同苹果品种和不同生长时期的表达差异。结果:P2’-GT cDNA全长1 452 bp,编码483 个氨基酸,相对分子质量53.6 kDa,等电点5.76,该基因编码蛋白是不稳定蛋白,不具有明显的跨膜结构,二级结构主要有α-螺旋、无规卷曲和延伸链组成,三级结构结果显示P2’-GT蛋白模型与对苯二酚葡萄糖基转移酶相似度最高(41.32%),进化分析表明P2’-GT与白梨糖基转移酶同源性最高。RTFQ-PCR分析发现P2’-GT在3 种苹果皮中均高效表达,在叶和根中微量表达,在果肉中不表达;P2’-GT的转录表达受苹果发育调控,在生长初期几乎不表达,随着苹果发育表达量逐渐增加,在生长中期达到最高值,随后开始减少,至苹果成熟期下降到最高值的50%水平。此外,P2’-GT在3 种苹果品种中表达不同,在澳洲青苹中表达量最高,在嘎啦中表达量最低。结论:本研究明确了P2’-GT的生物信息学特性及P2’-GT基因在不同生长期和不同部位的表达差异,为调控根皮苷合成的研究提供理论依据。

关键词: 根皮苷, 根皮苷-2-O-糖基转移酶, 克隆, 生物信息学, 实时荧光定量聚合酶链式反应

Abstract: Objective: Phloridzin 2-O-glucosyltransferase (P2’-GT) is the key enzyme involved in the final step in the phloridzin biosynthetic pathway, catalyzing transformation of phloretin into phlorizin. The purpose of this study was to clone a P2’-GT gene from Fuji apple and to investigate bioinformatics analysis of the protein encoded by the gene and its expression pattern. Methods: RNA was extracted from apple peel and was used to synthesize a cDNA template by reverse transcription. Specific primers were designed based on the P2’-GT reporter gene for PCR amplification and sequencing. Bioinformatic analyses were performed using online software (Pro Param tool and TMHMM). The differential transcript levels of P2’-GT in different organs of apple varieties at different growth stages were detected using real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR). Results: The cDNA was 1 452 bp long, encoding 483 amino acid residues. The encoded protein was unstable with a molecular mass of 53.6 kDa and a theoretical PI of 5.76. In addition, the protein had no obvious transmembrane structure. Its secondary structure consisted of α-helix, random coil and extended strand. The protein tertiary structure exhibited high similarity to hydroquinone glucosyltransferase (41.32%). Phylogenetic analysis indicated that P2’-GT had the highest homology to Pyrus bretschneideri glucosyltransferase. The expression level of this gene was high in apple peel but extremely low in leaves and roots and zero in apple pulp. P2’-GT gene expression was related to the growth period; transcript level was rarely detectable in the early stage, but increased rapidly to a maximum in the middle stage and then decreased by 50% in the maturation stage. Significant differences in the transcript level of this gene were found among three different varieties, with the highest and lowest level being observed in Granny Smith and Gala, respectively. This research provides theoretical support for the role of P2’-GT genes in regulating the synthesis of phloridzin.

Key words: phloridzin, phloridzin 2’-O-glycosyltransferase, cloning, bioinformatics, real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR)

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