大花君子兰查尔酮合酶基因CmCHS的克隆及其功能验证

doi:10.16420/j.issn.0513-353x.2021-0090

摘要/Abstract

摘要：

为研究大花君子兰（Clivia miniata）查尔酮合酶的结构及功能,基于前期构建的大花君子兰转录组数据库,对参与花青素合成的CHS基因家族进行鉴定和筛选。在大花君子兰中克隆得到4个CmCHS（CmCHS1 ~ CmCHS4）,其开放阅读框全长分别为1 173、1 170、1 173和1 173 bp,编码390、389、390、390个氨基酸。氨基酸序列比对分析证实,CmCHS具备该基因家族典型的保守结构域。进化树分析结果显示,4条CmCHS属于真实的CHS,编码蛋白属于Ⅲ型聚酮合成酶（PKS）,亚细胞定位于细胞质和细胞核中。基因表达特异性分析结果表明,CmCHS1和CmCHS2在花朵初开和盛开期表达量较高;CmCHS1、CmCHS2、CmCHS3在红色叶片和果皮中的表达量大大高于绿色叶片和果皮,CmCHS4则相对较低。构建原核表达载体pET-32-CmCHS,体外酶活检测表明,4个CmCHS重组蛋白皆可催化香豆酰辅酶A和丙二酰辅酶A生成柚皮素查尔酮。

关键词: 君子兰, 类黄酮, 花青素, 查尔酮合酶, 酶活

Abstract:

In order to investigate the structure and function of Chalcone Synthase from Clivia miniata,members of CHS gene family were screened and identified based on the previously constructed transcriptome database. The results showed that four CmCHSs（CmCHS1,CmCHS2,CmCHS3 and CmCHS4）genes were cloned in C. miniata. The open reading frames of the four CmCHSs were 1 173,1 170,1 173 and 1 173 bp and encoding 390,389,390,390 respectively. Amino acid sequence that CmCHSs had typical the conserved domains. Phylogenetic tree analysis showed that the four CmCHS genes clustered with the bona-fide CHSs from other plants and belonged to type Ⅲ polyketide synthase（PKS）super family. The results of subcellular localization showed that they were localized in the cytoplasm and nucleus. Gene expressionanalysis showed CmCHS1 and CmCHS2 were highly expressed at the late stage of flower development process,and the transcripts of CmCHS1,CmCHS2 and CmCHS3 were confirmed to the extensively detected in pigmented leaves and red fruit peels. In contrast,the expression level of CmCHS4 gene was relatively low. In order to firmly verified the roles of CmCHSs in the biosynthesis of anthocyanins,prokaryotic expression vectors,pET-32-CmCHS,were constructed and recombinant proteins were prepared and purified,which were subjected to in vitro enzyme activity analysis. Results showed that the four CmCHS recombinant proteins could catalyze coumarinyl-CoA and malonyl-Coenzyme A into naringenin chalcone. This study explored the bioinformatics analysis,gene expression analysis,and functional analysis of CmCHS genes,and laid a theoretical foundation for the genetic transformation and flower color improvement of C. miniata.

Key words: Clivia miniata, flavonoid, anthocyanin, chalcone synthase, enzyme activity

中图分类号:

S682.13

刘玥, 李月庆, 孟祥宇, 黄靖, 汤昊, 李源恒, 高翔, 赵春莉. 大花君子兰查尔酮合酶基因CmCHS的克隆及其功能验证[J]. 园艺学报, 2021, 48(10): 1847-1858.

LIU Yue, LI Yueqing, MENG Xiangyu, HUANG Jing, TANG Hao, LI Yuanheng, GAO Xiang, ZHAO Chunli. Cloning and Functional Characterization of Chalcone Synthase Genes（CmCHS）from Clivia miniata[J]. Acta Horticulturae Sinica, 2021, 48(10): 1847-1858.

图/表 6

表1 本研究中的引物序列

Table 1 Primer sequences in this study

用途 Use	基因 Gene	引物序列（5′-3′） Primer sequence
基因ORF的克隆 Cloning the full-length of ORF	CmCHS1	F:ATGGTGAGTATCGATGAGATT;R:CATCGCGCCGATGATTAATT
	CmCHS2	F:ATGGTGAGTGTCCATGAGATTCGC;R:CACGATGTGTCGCATCGATAAATT
	CmCHS3	F:ATGGTGAGCGTGGAAGAGATC;R:GGTATCACACCGATGATTAATT
	CmCHS4	F:ATGGCCACAATTGAGGAGATCAG;R:ACGCTTCACACCGCTAGTTAACT
	T7	F:TAATACGACTCACTATAGGG;R:GCTAGTTATTGCTCAGCGG
实时荧光定PCR Quantitative Real-time PCR	CmCHS1	F:GACATGCCCGGTGCAGACTACCA;R:AGCCGTGATCTCCGAGCAGACG
	CmCHS2	F:GACGAGACAAGTGTTGAGCGAGTA;R:TCCAGGTCCGAATCCAAAGAGC
	CmCHS3	F:AGGAGCGATAGACGGGCACTTA;R:GAACAGCGAGTTCCAATCTAACATC
	CmCHS4	F:GGAGGTGCTGAAGGAGTATGGGAACA;R:TCCAGGGCCGAACCCAAATAGA
	CmActin	F:GCATCACACCTTCTACAA;R:CATTGTAGAAGGTGTGATG
制备重组蛋白 Preparation of recombinant protein	CmCHS1	F:TGGCTGATATCGGATCCATGGTGAGTATCGATGAG; R:CGACGGAGCTCGAATTCTTAATTAGTAGCCGCGCT
	CmCHS2	F:TGGCTGATATCGGATCCATGGTGAGTGTCCATGAG; R:CGACGGAGCTCGAATTCTTAAATAGCTACGCTGTG
	CmCHS3	F:TGGCTGATATCGGATCCATGGTGAGCGTGGAAGAG; R:CGACGGAGCTCGAATTCTTAATTAGTAGCCACACT
	CmCHS4	F:TGGCTGATATCGGATCCATGGCCACAATTGAGGAG; R:CGACGGAGCTCGAATTCTCAATTGATCGCCACACT
亚细胞定位 Subcellular	GFP(35S:GFP)	F:CTGATTACGCTCATATGATGGTGAGCAAGGGCGAG; R:AGGATTCAATCTTAAGTTACTTGTACAGCTCGTCCATG
localization	(GFP)CmCHS1	F:GACGAGCTGTACAAGCATATGGTGAGTATCGAT; R:CAACAGGATTCAATCTTAAGTTAATTAGTAGCCGC
	(GFP)CmCHS2	F:GACGAGCTGTACAAGCATATGGTGAGTGTCCAT; R:CAACAGGATTCAATCTTAAGTTAAATAGCTACGCT
	(GFP)CmCHS3	F:GACGAGCTGTACAAGCATATGGTGAGCGTGGAA; R:CAACAGGATTCAATCTTAAGTTAATTAGTAGCCAC
	(GFP)CmCHS4	F:GACGAGCTGTACAAGCATATGGCCACAATTGAG; R:CAACAGGATTCAATCTTAAGTCAATTGATCGCCAC

图1 君子兰（Clivia miniata）与其他物种CHS序列比对 AtCHS:拟南芥,AAA32771.1;FhCHS:香雪兰,JF732897;GbCHS:银杏,AAT68477.1;ZmCHS:玉米,CAA42763.1;MsCHS:紫花苜蓿,P30074.1;VvCHS:葡萄,BAA31259.1;VvSTS:葡萄,ABV82966.1。黄色框表示CHS中3个守恒催化残基;绿框中氨基酸决定CHS底物特异性;红色框中氨基酸为丙二酰辅酶A的结合区域;黑色矩形框为CHS的高度保守结构域;蓝色圆圈表示与香豆素酰辅酶A结合过程中重要的残基以及CHS环化反应的特异性残基。

Fig. 1 Sequence alignment of CHSs from Clivia miniata and other species AtCHS:Arabidopsis thaliana,AAA32771.1;FhCHS:Freesia hybrida,JF732897;GbCHS:Ginkgo biloba,AAT68477.1;ZmCHS:Zea mays,CAA42763.1;MsCHS:Medicago sativa,P30074.1;VvCHS:Vitis vinifera,BAA31259.1;VvSTS:Vitis vinifera,ABV82966.1.The yellow box represents three conserved catalytic residues in CHS. The green frame amino acid determines the specificity of CHS substrate;The red frame amino acid is the binding region of malonyl coenzyme A. The black rectangular box indicates the highly conserved domains of CHS. The blue circles represent important residues in binding to coumarinyl coenzyme A and residues specific to the cyclic reaction of CHS.

图2 CmCHS系统进化树数字表示自展值。

Fig. 2 Phylogenetic analysis of CmCHSs Numbers indicate bootstrap values.

图3 CmCHS 亚细胞定位结果将4个重组质粒瞬时转染到野生型拟南芥原生质体中,在室温黑暗中培养21 h。激光扫描共聚焦显微镜捕获荧光蛋白。

Fig. 3 Subcellular localization of CmCHSs Four recombinant plasmids were transiently transfected into wild-type Arabidopsis protoplasts,and incubated for 21 h at room temperature in the dark. Laser scanning confocal microscopy captured the fluorescent proteins.

图4 君子兰CmCHS在不同阶段（A）、不同颜色果皮（B）和叶片（C）中的表达

Fig. 4 CmCHS expressions in different colored tissues and stages of Clivia miniata

图5 CmCHS1、CmCHS2、CmCHS3和CmCHS4的体外酶学分析 A:提取重组蛋白SDS-PAGE;B:HPLC。

Fig. 5 In vitro enzymatic analysis of CmCHS1,CmCHS2,CmCHS3 and CmCHS4 A:The recombinant protein was extracted from sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）;B:HPLC.

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