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中国农学通报 ›› 2016, Vol. 32 ›› Issue (1): 118-124.doi: 10.11924/j.issn.1000-6850.casb15070012

所属专题: 生物技术

• 林学 园艺 园林 • 上一篇    下一篇

桂花查耳酮合酶chs 基因的克隆及序列分析

徐秀荣,马 燕,臧德奎   

  1. 山东农业大学林学院,山东农业大学林学院,山东农业大学林学院
  • 收稿日期:2015-07-02 修回日期:2015-07-25 接受日期:2015-08-04 出版日期:2016-01-28 发布日期:2016-01-28
  • 通讯作者: 臧德奎
  • 基金资助:
    山东省自然科学基金(ZR2013CQ008)。

Cloning and sequencing analysis of Sweet Solive chalcone synthase gene

  • Received:2015-07-02 Revised:2015-07-25 Accepted:2015-08-04 Online:2016-01-28 Published:2016-01-28

摘要: 克隆与桂花(Osmanthus fragrans)花色形成相关基因查耳酮合酶(Ofchs),并对该基因的序列特征、进化关系进行分析,为探究该基因在桂花花色形成中的功能提供理论依据。以桂花‘丹桂’品种为试材,采用RT-PCR和RACE方法克隆丹桂查耳酮合酶(chalcone synthase, chs)基因cDNA全长。利用NCBI和DNAMAN分析软件,对该基因进行核苷酸序列以及氨基酸序列的分析,利用DNAMAN软件直接构建系统进化树,以分析该基因在不同物种间的进化关系。结果表明:从‘丹桂’花瓣中成功获得了1 个丹桂查耳酮合酶基因cDNA全长,该基因全长为1383 bp,包含48 bp 的5′非编码区、162 bp 的3′非编码区和1个长度为1173 bp 编码390 个氨基酸的开放阅读框。序列分析表明,该基因具有Cysl64、His303和Asn336 3个活性位点及2 个决定底物特异性的苯丙氨酸残基Phe215、Phe265。该基因也具有查尔酮合酶家族的2 条特征多肽序列RFMMYQQGCFAGGTVLR和GVLFGFGPGL。Blast 序列分析显示,该基因与同属木犀科的油橄榄相似性达95%,与其他双子叶植物的相似性达72%~76%。克隆出的丹桂chs 基因属于查尔酮合酶家族,系统进化分析表明,该基因聚类具有明显的种属特性,桂花与唇形科等植物的chs 亲缘关系较近,相似性达76%以上,符合植物分类学分类,为今后探究该基因在花色行形成中的作用机制奠定了基础。

关键词: 干旱胁迫, 干旱胁迫, 锦鸡儿属, 抗旱性, 隶属函数

Abstract: The full-length cDNA of chs gene of Osmanthus fragrans was cloned which was related to color formation, the characteristics of the gene sequence and evolution relationships were analyzed to provide a theoretical basis for exploring gene function in the form of color. In this study, a full-length cDNA sequence of chs gene was obtained from Osmanthus fragrans by using RT-PCR and RACE. The gene nucleotide and amino acid sequences were analyzed by using NCBI and DNAMAN and a phylogenetic tree was constructed by using DNAMAN software directly, which would help analyze the gene evolutionary relationships among different species. Result showed that: the full-length gene was cloned from Osmanthus fragrans successfully, which was displayed as Osmanthus fragrans chs gene, which was 1383 bp in full length and included 5′URT of 48 bp and 3′ URT of 162 bp, had an open reading frame of 1173 bp encoding a deduced polypeptide of 390 amino acids. Sequence analysis revealed that this gene had 3 active sites: Cysl64, His303 and Asn336, and 2 decisions substrate specificity of phenylalanine residues: Phe215 and Phe265. Ofchs had 2 characteristics of polypeptide sequence of chs synthase superfamily: RFMMYQQGCFAGGTVLR and GVLFGFGPGL. Blast sequence showed that similarity between Osmanthus fragrans and Olea europaea was up to 95% and was at around 72%-76% with the other dicots. Ofchs gene belonged to chs family, phylogenetic analysis showed that the gene cluster had the distinct species characteristics, Osmanthus fragrans had a near relationship to Labiatae, which was up to 76%, and it was in line with plant taxonomy, and laid the foundation to explore the mechanism of Ofchs in the formation of the color line for the future.

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