中国中药杂志

2019, v.44(09) 1799-1807

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异叶天南星查尔酮合成酶和异构酶基因的克隆及蛋白的结构性质分析
Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum

张声祥;施圆圆;王晨凯;赵德蕊;杨青山;马克龙;吴家文;
ZHANG Sheng-xiang;SHI Yuan-yuan;WANG Chen-kai;ZHAO De-rui;YANG Qing-shan;MA Ke-long;WU Jia-wen;Graduate School,Anhui University of Chinese Medicine;Key Laboratory of Xin'an Medicine,Ministry of Education,Anhui University of Chinese Medicine;College of Pharmacy,Anhui University of Chinese Medicine;Synergetic Innovation Center of Anhui Authentic Chinese Medicine Quality Improvement;Anhui Academy of Chinese Medicine;Clinical College of Integrated Traditional Chinese and Western Medicine,Anhui Unive

摘要(Abstract):

查尔酮合成酶(CHS)和查尔酮异构酶(CHI)是黄酮类物质生物合成途径中的关键酶,该研究从中药异叶天南星(Arisaema heterophyllum Blume,Ah)转录组数据库中筛选出查尔酮合成酶基因(Ah CHS)和查尔酮异构酶基因(Ah CHI),利用RT-PCR技术克隆了它们的开放阅读框(ORF)部分,并对其编码蛋白Ah CHS和Ah CHI的理化性质、表达及结构特点进行分析。Ah CHS的ORF长度为1 176 bp,编码392个氨基酸的蛋白质;而Ah CHI的ORF长度为630 bp,编码蛋白含209个氨基酸。Ah CHS作为对称同二聚体,N端螺旋互相交换,每个单体由2个亚结构域组成,位于2个亚结构域之间交界处的4个保守的氨基酸残基构成了酶的活性中心; Ah CHI的三级结构采用了开放的β-三明治褶皱,1个大的β折叠片层(β4~β11)和1个α螺旋片层(α1~α7)组成其核心结构,其中β4,β5,α4和α6在不同物种间高度保守,部分疏水氨基酸形成了酶的活性位点。系统进化树分析发现Ah CHS与火鹤花CHS亲缘关系最近;而Ah CHI与紫牡丹CHI亲缘关系最近。表达分析表明编码Ah CHS的基因在根和块茎中的表达量明显比叶多,而编码Ah CHI的基因在块茎部位的表达量最高。该研究为Ah CHS和Ah CHI功能的进一步研究及植物黄酮类化合物生物合成途径的解析提供了基础。
Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced β-sandwich fold. A large β-sheet( β4-β11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning β4,β5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

关键词(KeyWords): 异叶天南星;基因克隆;查尔酮合成酶;查尔酮异构酶;生物信息学分析
Arisaema heterophyllum;gene cloning;chalcone isomerase;chalcone synthase;bioinformatics analysis

Abstract:

Keywords:

基金项目(Foundation): 中央本级重大增减支项目(2060302);; 安徽高校自然科学研究重大项目(KJ2018ZD028);; 安徽省自然科学基金项目(1608085MH177);; 国家级大学生创新训练项目(201810369050);; 安徽省留学人员科技活动启动项目(20151x024)

作者(Authors): 张声祥;施圆圆;王晨凯;赵德蕊;杨青山;马克龙;吴家文;
ZHANG Sheng-xiang;SHI Yuan-yuan;WANG Chen-kai;ZHAO De-rui;YANG Qing-shan;MA Ke-long;WU Jia-wen;Graduate School,Anhui University of Chinese Medicine;Key Laboratory of Xin'an Medicine,Ministry of Education,Anhui University of Chinese Medicine;College of Pharmacy,Anhui University of Chinese Medicine;Synergetic Innovation Center of Anhui Authentic Chinese Medicine Quality Improvement;Anhui Academy of Chinese Medicine;Clinical College of Integrated Traditional Chinese and Western Medicine,Anhui Unive

DOI: 10.19540/j.cnki.cjcmm.20190130.001

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