藤茶查尔酮合成酶基因AgCHS1的克隆及功能鉴定

doi:10.3864/j.issn.0578-1752.2020.24.012

摘要/Abstract

摘要：

【目的】查尔酮合成酶（chalcone synthase,CHS）是植物类黄酮生物合成的第一个关键酶,从藤茶中克隆AgCHS1,分析其序列特征及其在藤茶中的组织表达特异性,通过体外酶活性检测和烟草遗传转化对其功能进行鉴定,为进一步研究藤茶类黄酮累积的调控机理提供理论基础。【方法】根据藤茶转录组测序结果设计引物,以藤茶叶片cDNA和基因组DNA为模板,PCR扩增得到AgCHS1。利用生物信息学方法分析该基因的序列特征,使用MEGA6和DNAMAN软件进行多重序列比对,并构建系统进化树。通过原核表达系统获得AgCHS1的重组蛋白,分析该重组蛋白对底物的催化活性,并通过高效液相色谱-质谱（HPLC-MS）对酶促反应产物进行鉴定。利用qRT-PCR技术对AgCHS1在藤茶不同器官的表达水平进行分析,并采用硝酸铝比色法测定相应的总黄酮含量变化。构建植物过量表达载体,通过叶盘法转化烟草,筛选阳性转基因后代,对T₂代株系花瓣中的花青素和黄酮醇含量进行检测。【结果】AgCHS1的ORF长1 182 bp,编码393个氨基酸,基因组序列长1 315 bp,含2个外显子和1个内含子。生物信息学分析表明,AgCHS1为稳定的亲水蛋白。通过与其他物种的CHS蛋白多序列比对发现,AgCHS1含有查尔酮合成酶家族的特征序列和活性位点残基,包括丙二酰CoA结合位点和三联活性中心位点,与其他物种的CHS序列一致性较高。系统进化分析显示,AgCHS1与葡萄、山葡萄的CHS处于同一进化分支,亲缘关系最近。荧光定量PCR结果表明,AgCHS1在成熟叶和花中的表达量最高,在老叶中的表达量最低。藤茶不同器官的总黄酮含量与AgCHS1表达水平呈显著正相关。体外酶活性分析显示,重组的AgCHS1蛋白可以催化底物对-香豆酰辅酶A和丙二酰辅酶A生成柚皮素,说明该蛋白具有查尔酮合成酶活性。获得5株烟草转基因阳性株系,其中2株的花瓣颜色明显加深;与对照相比,转基因株系OE3、OE4花瓣中的花青素含量分别提高56.6%和25.3%,OE3的黄酮醇含量没有显著差异,OE4的黄酮醇含量提高39.1%。【结论】AgCHS1是藤茶中催化合成查尔酮的关键酶,过量表达AgCHS1可以提高转基因植物中花青素和黄酮醇的含量。

关键词: 藤茶, 查尔酮合成酶, 酶活性, 花青素, 黄酮醇

Abstract:

【Objective】Chalcone synthase(CHS) is the first key enzyme in the biosynthesis of plant flavonoids. In this study, the AgCHS1 was cloned from Ampelopsis grossedentata, the gene sequence and tissue specific expression were analyzed, and the gene function was characterized via in vitro enzymatic activity assay and transformation of N. tabacum, aiming to provide a theoretical basis for further elucidating the flavonoid accumulation of the A. grossedentata. 【Method】The primers were designed according to the transcriptome of A. grossedentata and the leaf cDNA and genomic DNA were used as templates to amplify the AgCHS1 with PCR. The sequence features were analyzed with bioinformatic methods. The MEGA6 and DNAMAN were used for multiple sequences alignment and phylogenetic tree construction. The AgCHS1 recombinant protein was obtained via prokaryotic expression, and the enzymatic activities of the protein on substrates were assayed by identifying the catalytic products with HPLC-MS. The expression of AgCHS1 in different organs was quantified by qRT-PCR, and the content of total flavonoids was determined by Al(NO₃)₃colorimetric method. The overexpression vector was constructed and transformed to N. tabacum by using the leaf disk method. The transgenic plants were screened and the contents of anthocyanin and flavonol in the petal of T₂ strain were measured.【Result】The ORF of AgCHS1 had a length of 1 182 bp and encoded 393 amino acids. The genomic DNA spanned 1 315 bp, containing 2 exons and 1 intron. The bioinformatic analysis showed that AgCHS1 was a stable hydrophilic protein. The results of sequence alignments indicated that the AgCHS1 possessed the signature sequence and enzymatic site residues of the chalcone synthase gene superfamily, including the binding site of malonyl-CoA and triple active center, which share a high similarity with the CHS proteins of other species. The phylogenetic analysis showed that AgCHS1 had a close relationship with and was in the same clade with the CHSs of Vitis vinifera and Vitis amurensis. The results of fluorescent qPCR showed that AgCHS1 expressed highest in the mature leaf and the flower, and lowest in the old leaf. There was a positive correlation between the expression level of AgCHS1 gene and the content of total flavonoids in different organs of A.grossedentata. The in vitro enzymatic analysis showed that the recombinant AgCHS1 was able to catalyze the substrates p-coumaroyl-CoA and CoA malonyl-CoA to produce naringenin, demonstrating the activity of chalcone synthase. 5 transgenic N. tabacum lines were obtained, 2 of which showed a significant deeper leaf color. The anthocyanin contented of the transgenic lines OE3 and OE4 increased by 56.6% and 25.3%, respectively, compared to the control. The flavonol content of line OE3 showed no significant difference, which of the line OE4 were increased by 39.1%.【Conclusion】The AgCHS1 was the key enzyme in the flavonoid biosynthesis of A. grossedentata, and the overexpression of AgCHS1 increased the contents of anthocyanin and flavonol in transgenic plants.

Key words: Ampelopsis grossedentata, chalcone synthetase, enzyme activity, anthocyanin, flavonol

许明,林世强,倪冬昕,伊恒杰,刘江洪,杨志坚,郑金贵. 藤茶查尔酮合成酶基因AgCHS1的克隆及功能鉴定[J]. 中国农业科学, 2020, 53(24): 5091-5103.

XU Ming,LIN ShiQiang,NI DongXin,YI HenJie,LIU JiangHong,YANG ZhiJian,ZHENG JinGui. Cloning and Function Characterization of Chalcone Synthase Gene AgCHS1 in Ampelopsis grossedentata[J]. Scientia Agricultura Sinica, 2020, 53(24): 5091-5103.

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引物名称 Primer name	序列（5'-3'） Primer sequence (5'-3')	用途 Purpose
AgCHS1-F	GGATCCATGGTGTCGGTGCAGGAAATCAGA	ORF序列扩增、载体构建
AgCHS1-R	GAGCTCTCAGTGAGCCAGTGGTGCAGAC	Amplification of ORF sequence and vector construction
AgCHS1-P1	ACTCCAGCCAACTGTGTCCA	转基因烟草的PCR鉴定
AgCHS1-P2	TGAGACCCACTTCACGCAA	PCR identification of transgenic tobacco
AgCHS1-qF	GCCTCAAACCCTCCGTCA	实时荧光定量PCR Quantitative real-time PCR
AgCHS1-qR	GACCCACGAGTGAATCCAAGT	Quantitative real-time PCR
NtEF1α-qF	AATTTTGACCAAGATCGACAGG	烟草内参基因 Control
NtEF1α-qR	CAGCAACAGTTTGACGCATG	Reference gene in tobacco
AgGAPDH-qF	CATCTCAGCCCCAAGCAA	藤茶内参基因
AgGAPDH-qR	GTGGCAGTAATGGAGTGAACAG	Reference gene in A.grossedentata