Cloning and Bioinformatics of Chalcone Synthase Gene of Moringa oleifera
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摘要:
目的 查尔酮合成酶是黄酮类生物合成途径中的第1个限速酶基因。从辣木中克隆查尔酮合成酶基因MoCHS1，并对其进行生物信息学分析，为进一步研究其生物学功能提供基础数据。 方法 根据NCBI数据库中的辣木基因组信息设计引物，以辣木叶片cDNA和基因组DNA为模板，PCR扩增获得MoCHS1基因序列。利用生物信息学方法分析其序列特征，使用DNAMAN9.0和MEGA10.0软件进行多重比对和构建系统进化树。 结果 MoCHS1 ORF序列长度为1 185 bp，编码394个氨基酸，基因组序列长1 387 bp，含有2个外显子和1个内含子。生物信息学分析表明，MoCHS1为稳定的亲水蛋白，以α-螺旋（45.43%）和不规则卷曲（31.98%）为主。MoCHS1含有查尔酮合成酶家族的保守序列和酶活性位点的关键氨基酸残基，包括7个环化袋氨基酸残基、3个辅酶A活性结合位点、半胱氨酸（Cys）-组氨酸（His）-天冬氨酸（Asn）三联体催化位点和查尔酮合成酶基因家族的2个高度保守的特征序列（RLMMYQQGCFAGGTVLR和GVLFGFGPGL），与其他物种的CHS 序列一致性较高。系统进化分析显示，MoCHS1与番木瓜聚在一类，说明其亲缘关系最近。 结论 成功分离了一个辣木查尔酮合成酶基因MoCHS1基因序列，该基因推测编码的氨基酸序列具有CHS家族蛋白的典型保守结构特征。研究结果有助于进一步研究辣木查尔酮合成酶基因的调控及基因家族进化机制和类黄酮合成调控机理。 Abstract:Objective The open reading frame and gDNA of the gene associated with the chalcone synthase (CHS), the first key enzyme in the flavonoid biosynthetic pathway in Moringa oleifera, were cloned for a bioinformatic analysis. Method Primers were designed according to the M. oleifera genome in NCBI database. The leaf cDNA and genomic DNA were used as templates to amplify the MoCHS1 with PCR for a subsequent bioinformatic analysis. DNAMAN 9.0 and MEGA 10.0 were used for the multiple sequence alignment and phylogenetic tree construction. Result The ORF of MoCHS1 was 1 185 bp encoded 394 amino acids. The genomic DNA spanned 1 387 bp containing 2 exons and 1 intron. MoCHS1 was a stable hydrophilic protein with a structure of 45.43% α helix and 31.98% random coil. The gene possessed conserved sequence as well as enzymatic site residues of the CHS gene superfamily, which included 7 amino acid residues of the cyclization pocket, the Cys-His-Asn catalytic triad sites, and the family signatures of CHSs (‘RLMMYQQGCFAGGTVLR’ and ‘GVLFGFGPGL’) sharing a high similarity with the CHS proteins of other species. The phylogenetic tree of MoCHS1 closely related to and in the same clade with the CHS of Carica papaya. Conclusion MoCHS1 was successfully isolated from M. oleifera sharing a typical conserved structure of the CHS gene family. The results provided crucial information for further study on the genes related to the flavonoid metabolic pathway as well as the expression regulation and evolution of the CHS gene family in M. oleifera. -
Key words:
- Moringa oleifera /
- flavonoid /
- chalcone synthase /
- bioinformatic analysis
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图 1 辣木MoCHS1基因的克隆与结构分析
注：A: MoCHS1的PCR扩增产物，M：DL2 000；1，2：以基因组DNA 为模板的扩增产物；3，4：以cDNA为模板扩增产物。B：MoCHS1基因的结构模式图
Figure 1. Cloning and structure of MoCHS1
of M. oleifera Note: A: PCR amplification product of MoCHS1; M: DL2000; 1 and 2: PCR amplification product of cDNA; 3 and 4: PCR amplification product of genomic DNA; B: structure of MoCHS1.
图 2 辣木MoCHS1 cDNA 核苷酸序列及推导的氨基酸序列
Figure 2. Nucleic acid and putative amino acid sequence of MoCHS1 of M. oleifera
图 3 MoCHS1的二级结构预测分析
注：蓝色：α-螺旋；紫色：无规则卷曲；红色：延伸链；绿色：β-转角
Figure 3. Prediction and analysis on secondary structure of MoCHS1
Note: Blue: α helix; purple: random coil; red: extended strand; green: β turn.
图 5 辣木MoCHS1氨基酸序列与其他物种CHS氨基酸序列的多序列比对分析
注：星号标出的三个氨基酸残基为辅酶A活性结合位点；箭头所示为环化袋残基位点；四方体所示三联体催化位点；下划线所示为查尔酮合成酶基因家族特征序列。
Figure 5. Multiple alignment on amino acid sequences of CHS of M. oleifera and other species
Note: Asterisks: active CoA-binding sites (Lys, Arg, and Lys); arrows: 7 amino acid residues of cyclization pocket including Thr, Met, Phe, Ile, Gly, Phe, and Pro sites; squares: catalytic triad sites of Cys, His, and Asn, and family signatures of CHSs (‘RLMMYQQGCFAGGTVLR’ and ‘GVLFGFGPGL’) are underlined.
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