目的 鉴定当归Angelica sinensis (Oliv.) Diels查耳酮异构酶（chalcone isomerase，CHI）基因家族成员，对其进行生物信息学分析、筛选候选基因并进行体外酶活实验，为进一步研究当归查耳酮异构酶在黄酮类化合物生物合成中的作用机制提供理论依据。方法 依据当归基因组数据，鉴定AsCHI基因家族成员，进行蛋白理化性质分析、二级结构预测、基因结构、启动子顺式作用元件以及系统进化分析，通过大肠杆菌异源表达和体外酶活验证基因功能，利用反转录实时荧光定量PCR（real-time quantitative polymerase chain reaction，RT-qPCR）分析其表达特征。结果 在当归基因组中共鉴定到5个AsCHIs，编码219～321个氨基酸，相对分子质量在24 000～36 000，理论等电点为4.75～9.11，二级结构以无规则卷曲和α-螺旋为主。启动子分析显示AsCHIs基因含有丰富的光响应元件、激素响应元件及非生物胁迫响应元件；系统进化分析将AsCHI1归类为I型CHI，AsCHI2为IV型CHI；AsCHI3～5属于III型CHI。体外酶活实验表明AsCHI1能够催化柚皮素查耳酮生成柚皮素；RT-qPCR结果显示AsCHI1和AsCHI2主要在当归的茎和叶中表达。结论 鉴定了5个AsCHIs家族成员，成功克隆了AsCHI1和AsCHI2，通过体外酶活实验验证了AsCHI1具有查耳酮异构酶活性。

Objective To systematically identify members of chalcone isomerase (CHI) gene family in Danggui (Angelica sinensis) and elucidate their roles in flavonoid biosynthesis. Methods Genome-wide screening was performed using the A. sinensis reference genome. Physicochemical parameters, secondary structures, gene structure, promoter cis-elements, and phylogenetic relationships of the identified AsCHIs were analyzed. Candidate genes were cloned, heterologously expressed in Escherichia coli, and assayed for in-vitro enzymatic activity. Expression profiles were quantified by RT-qPCR. Results Five AsCHI genes encoding 219—321 amino acids (Mw 24 000—36 000; pI 4.75—9.11) were identified. Their predicted secondary structures are dominated by α-helices and random coils. Promoter regions showed abundant light-, hormone- and stress-responsive elements. Phylogenetic analysis classified AsCHI1 in the type-I CHI clade, AsCHI2 in type-IV, and AsCHI3-5 in type-III. Recombinant AsCHI1 catalyzed the conversion of naringenin chalcone to naringenin in vitro, confirming CHI activity. Transcript analysis revealed that AsCHI1 and AsCHI2 are predominantly expressed in stems and leaves. Conclusion Five members of the AsCHI family were identified, with AsCHI1 and AsCHI2 successfully cloned. In vitro enzyme activity assays confirmed that AsCHI1 possesses chalcone isomerase activity.

R286.12

云南省科技计划项目（202502AS100012，202304B1090009）；云南特色植物提取实验室自主研究项目基金（2022YKZY001）；云南省兴滇英才支持计划“青年人才”项目（XDYC-QNRC-2022-0219）