茶叶中苯乙腈的创伤诱导合成途径及香气影响研究

Phenylacetonitrile (PAN) is one important volatile emitted by tea plant (Camellia sinensis), in response to attack by insects. PAN was also determined to be one typical volatile for oolong tea. The biosynthesis of this volatile is triggered by leaf wound induced by herbivores or green-making process. Nevertheless, the biosynthetic pathway, catalytic enzyme, and gene regulation mechanism of PAN remain unknown for tea plant. Impact of PAN on perceived aroma of oolong tea would also be interesting. In this study, stable isotope labeling is applied to investigate the biosynthetic pathway of PAN, when induced by Ectropis oblique hypulina feeding or green-making treatment. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is utilized to determine the endogenous precursors of PAN. Bioinformatics tools, rapid amplification of cDNA ends (RACE), as well as quantitative real-time PCR techniques are used to the clone, sequences analysis, and expression level screening of CYP71s/CYP79s homologous genes. Enzyme assay coupled with gas chromatography-mass spectrometer (GC-MS) is further applied to determine the CYP enzyme catalyzing the biosynthesis of PAN. The impact of PAN on oolong tea aroma is evaluated by estimation of flavor dilution (FD) factor and odor active value (OAV), together with quantitative sensory analysis. Our work will contribute to the biosynthesis investigation of herbivore-induced volatiles, as well as processing chemistry of tea aroma. A better knowledge of CYP genes involving in tea plant is also expectable.

苯乙腈是茶树受虫害创伤后释放的重要化学信息物质，也是茶鲜叶做青损伤而诱导合成的典型挥发物，在乌龙茶中稳定存在且相对含量较高。然而，茶叶中苯乙腈的创伤诱导合成途径、关键催化酶和表达调控基因尚未探明，其对乌龙茶香气品质的影响也未知晓。本项目采用稳定同位素标记法，探明茶叶受茶尺蠖取食、做青损伤而释放苯乙腈的生物合成路径，利用UPLC-MS/MS对其内生的前体物质进行鉴定。采用生物信息学手段、cDNA末端快速扩增技术、qRT-PCR等，对茶树中CYP71/CYP79家族同源基因进行克隆、序列分析和表达量筛选，进一步结合酶反应分析对目标基因进行功能鉴定，以明确茶叶中苯乙腈诱导合成途径的关键催化酶及表达调控基因。通过感官定量描述分析、GC-O嗅辨分析和FD/OAV值测算，系统性评价苯乙腈对乌龙茶香气品质的影响。项目研究成果对于揭示茶叶创伤诱导挥发物的合成代谢机理、茶树CYP家族基因的功能有重要意义。

（一）项目背景、科学意义.苯乙腈（phenylacetonitrile, PAN）是一种具有联系“茶树虫害胁迫—加工机理—香气化学”特质的物质。了解苯乙腈对乌龙茶香气品质的影响，有助于丰富茶叶的香气化学理论。阐明苯乙腈的创伤诱导挥发物的合成机理，有助于了解乌龙茶加工过程中特定香气成分产生的原因和影响因素。苯乙腈生物合成途径中关键催化酶及表达调控基因的鉴定，可为茶树CYP基因的功能挖掘提供参考。.（二）主要研究内容.a. 揭示茶叶中苯乙腈的创伤诱导合成途径，明确其前体物质；b. 鉴定苯乙腈诱导合成途径的关键酶及调控基因；c. 探明苯乙腈的呈香活性及对乌龙茶香气品质的影响。.（三）重要结果、关键数据.1. 苯乙腈在铁观音茶样第一泡茶汤中的平均浸出率为32.2±9.3%，平均浓度为57.52±16.18 μg/L。苯乙腈在热茶汤中的气味阈值为16.44 μg/L，在冷茶汤中的气味阈值为13.62 μg/L。苯乙腈的呈香活性值（OAV）为3.5。添加了阈值苯乙腈的铁观音茶汤香型表现为发酵感、木香显著增强，果香显著减弱。.2.外源添加L-[2H8]苯丙氨酸后，叶片中有被同位素标记的苯乙腈的生成，验证了在茶叶中苯丙氨酸是苯乙腈的合成前体物质。苯乙腈的释放量随摇青次数的增加而增加；成熟叶中的苯乙腈释放量显著高于幼叶和顶芽，顶芽中未能检测到苯乙腈的释放。.3. 通过氨基酸序列的同源比对，得到5条茶树CYP71/CPY79候选基因。在‘黄观音’叶片中成功克隆得到苯乙腈合成相关基因CsCYP79A1基因，且该基因在适制乌龙茶的茶树品种中的表达量更高，随着摇青次数的增加，表达量先上升后下降，且在成熟叶片中的表达量显著高于幼叶和顶芽，为苯乙腈诱导合成途径的关键酶及调控基因之一。