忍冬（Lonicera japonica）是中药金银花和忍冬藤的基原植物，含有大量酚酸类和黄酮类化合物。其中最具代表性的绿原酸和木犀草苷都是苯丙氨酸经过多步级联反应产生。两种BAHD酰基转移酶：莽草酸/奎宁酸羟基肉桂酰转移酶（Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase，HCT）和奎宁酸羟基肉桂酰基转移酶（Hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase，HQT）是生成绿原酸的关键酶。尿苷二磷酸糖基转移酶（UDP-glycosyltransferase，UGT）是糖基化修饰类黄酮并赋予其结构多样化、稳定性及水溶性的关键修饰酶。本研究通过挖掘忍冬基因组数据，分析了忍冬中BAHD酰基转移酶超家族及UGT家族成员，并结合基因在不同组织部位、不同发育时期的表达模式，蛋白原核表达及体外酶活测定，植株不同部位化合物含量测定等实验，揭示了忍冬绿原酸生物合成关键酶基因功能差异的分子机制及相应的生理功能。主要研究内容和实验结果如下： 1.忍冬BAHD基因超家族成员分析：忍冬基因组共鉴定120个BAHD酰基转移酶基因，依据Kruse分类原则，可分为6个进化分支。其中，Clade 5包含1个HCT和2个HQT。 2.不同组织部位咖啡酰奎宁酸类（caffeoylquinic acids，CQAs）含量与LjHQT1/2、LjHCT表达相关性研究：对忍冬组培苗不同组织部位（根、茎、叶）及田间多年生忍冬花不同发育时期（S1-S6）中CQAs化合物含量进行UPLC测定，同时通过qRT-PCR分析LjHQT1/2及LjHCT表达量差异。结果表明，忍冬CQAs的合成与LjHQT1和LjHCT的表达正相关，LjHQT2的表达模式与LjHQT1存在明显差异，可能行使不同功能或受不同因素调控。 3.LjHQT1/2和LjHCT对低温的响应：对忍冬组培苗分别进行25℃（对照）和10℃（低温胁迫）培养，并对无根苗中CQAs的含量及LjHQT1/2、LjHCT的表达量变化进行测定。结果表明，低温处理后忍冬组培苗的CQAs积累增加，与LjHQT1和LjHCT的基因表达变化趋势一致，表明LjHQT1和LjHCT能够响应低温胁迫。LjHQT2的表达量在低温胁迫后降低，与其他两个基因存在明显的功能差异，可能负责介导绿原酸合成响应其他生理过程。 4.忍冬LjHQT1/2和LjHCT蛋白的表达及功能分析：通过原核表达体系获得的重组蛋白为可溶性。体外酶促反应结果显示，三个蛋白均能催化咖啡酰CoA与奎宁酸反应生成绿原酸，LjHQT1/2的催化产物还包括隐绿原酸。此外，三个蛋白均能催化p-香豆酰CoA与奎宁酸生成3-p-香豆酰奎宁酸和4-p-香豆酰奎宁酸。当酰基受体为莽草酸时，LjHCT可催化生成5-咖啡酰莽草酸或4-p-香豆酰莽草酸和5-p-香豆酰莽草酸。测定酶动力学相关参数，结果表明，在酰基供体（咖啡酰CoA或p-香豆酰CoA）饱和时，LjHQT2相较于LjHQT1对奎宁酸显示出更高的亲和力，而LjHQT1无论底物是咖啡酰CoA还是p-香豆酰CoA，其催化效率都高于LjHQT2。亚细胞定位结果显示，LjHQT1、LjHQT2与LjHCT均在胞质表达。 5.忍冬UGT基因家族的鉴定及LjUGT197、LjUGT198的克隆及表达：从忍冬基因组数据中共鉴定到241个UGT基因家族成员，可分为17组（A-P，R）。基于序列相似性并结合忍冬花发育6个时期的转录组及代谢组数据，筛选得到2个UGT，根据染色体定位信息分别命名为LjUGT197、LjUGT198。通过原核表达体系成功诱导并纯化得到可溶性重组蛋白。 本研究建立了忍冬无菌苗继代培养体系，首次完成无菌苗根、茎、叶中绿原酸类化合物种类和含量测定；首次揭示LjHQT2基因在忍冬不同部位及花不同发育时期的表达模式，并通过体外酶活实验证明LjHQT2蛋白也具有生成绿原酸的功能。此外，鉴定了2个可能参与黄酮类化合物合成的忍冬UGT基因，并进行了初步的体外蛋白表达研究。本研究为全面阐释绿原酸在忍冬发育和逆境胁迫中的生理功能提供了实验证据，为挖掘参与黄酮糖苷化合物合成的UGT奠定了基础。

Lonicera japonica, the original plant of the traditional Chinese medicines "Jinyinhua" and "Rendongteng", contains abundant phenolic acids and flavonoids. The representative compounds chlorogenic acid (CGA) and luteoloside are biosynthesized through multi-step cascade reactions from phenylalanine. Two BAHD acyltransferases, hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT), serve as key enzymes in CGA biosynthesis. UDP-glycosyltransferases (UGTs) play crucial roles in flavonoid glycosylation, enhancing structural diversity, stability, and water solubility. This study investigated the molecular mechanisms underlying functional differentiation of key enzymes in CGA biosynthesis through genome-wide identification of BAHD superfamily and UGT family members in L. japonica, combined with gene expression profiling across tissues and developmental stages, prokaryotic protein expression, in vitro enzymatic assays, and phytochemical quantification. The main findings are as follows:

BAHD superfamily identification: 120 BAHD acyltransferase genes were identified in L. japonica, classified into six clades following Kruse's classification. Clade 5 include1 HCT and 2 HQT.

Tissue-specific caffeoylquinic acids (CQAs) accumulation and gene expression correlation: UPLC quantification revealed differential CQAs contents in various tissue cultures (roots, stems, leaves) and developmental flower stages (S1-S6), while qRT-PCR analysis demonstrated positive correlations between CQAs biosynthesis and LjHQT1/LjHCT expression. LjHQT2 exhibited distinct expression patterns, suggesting functional divergence.

Low-temperature responses: Under cold stress (10℃ vs control 25℃), increased CQAs accumulation paralleled upregulated LjHQT1 and LjHCT expression. LjHQT2 expression decreased, indicating functional differentiation in mediating stress responses.

Enzymatic characterization of LjHQT1/2 and LjHCT: Recombinant proteins expressed in E. coli demonstrated catalytic activities. All three enzymes converted caffeoyl-CoA and quinate to CGA, with LjHQT1/2 additionally producing cryptochlorogenic acid. p-Coumaroyl-CoA could be converted to 3/4-p-coumaroyl quinate. LjHCT catalyzed formation of 5-caffeoyl shikimate or 4-p-coumaroyl shikimate and 5-p-coumaroyl shikimate using shikimate as acceptor. Kinetic analysis revealed LjHQT2's higher affinity for quinate, while LjHQT1 showed superior catalytic efficiency regardless of acyl donor. Subcellular localization confirmed that LjHQT1, LjHQT2 and LjHCT are all cytoplasmic expression.

UGT family identification and characterization: 241 UGT genes classified into 17 groups (A-P, R) were identified. LjUGT197 and LjUGT198 were cloned based on sequence similarity, transcriptomic and metabolomic data from flower development stages, with soluble recombinant proteins successfully expressed.

This study established an in vitro propagation system for L. japonica and first reported tissue-specific CQAs profiles. It revealed LjHQT2's differential expression patterns and catalytic capability, and identified two flavonoid-related UGT candidates. These findings provide experimental evidence for understanding CGA's physiological roles in development and stress responses, while laying foundation for exploring UGT-mediated flavonoid glycosylation.