醇类芳香物质是果实品质的重要组成，根据结构可分为直链类、萜类、苯环类等。醇类芳香物质可以通过萜类、脂肪酸和氨基酸等途径合成。不同果实中的醇类芳香物质组分不同，来源于萜类途径的E-香叶醇是柑橘果实的主要醇类芳香物质，番茄果实中的醇类芳香物质则主要来自脂肪酸途径的C5和C6醇。以柑橘和番茄果实为材料，围绕萜类和脂肪酸途径的醇类芳香物质，开展结构基因鉴别和转录因子筛选研究。主要结果如下： 1.基于体外原核表达和同源瞬时表达技术，鉴别出了参与E-香叶醇生物合成的结构基因CitTPS16。利用甜橙基因组数据库，开展了萜类合成酶(TPS)基因克隆，构建了10个TPS重组蛋白并分析了酶活性，发现属于TPS-b亚家族的CitTPS16可以体外催化香叶基焦磷酸(GPP)形成E-香叶醇。据此，在柑橘叶片和果实中分别进行瞬时过量表达分析，发现过表达CitTPS16显著促进了E-香叶醇含量的增加。可见CitTPS16参与了E香叶醇生物合成，为开展转录调控研究提供了靶标基因。 2.基于烟草双荧光素酶、酵母单杂和EMSA检测技术，鉴别出了转录激活结构基因CitTPS16表达的转录因子CitERF71。利用甜橙基因组数据库，克隆获得了48个AP2／ERF家族成员。烟草双荧光素酶分析显示，CitERF71转录激活CitTPS16的启动子活性。在柑橘果实发育阶段过程中，CitERF71转录本含量分别与结构基因CitTPS16表达水平(r= 0.79，P<0.05)、E-香叶醇含量(r=0.96，P<0.05)呈正相关关系。酵母单杂和EMSA实验结果表明，CitERF71可以直接结合CitTPS16启动子的ACCCGCC和GGCGGG序列。研究认为CitERF71可通过转录激活CitTPS16启动子，调控柑橘果实醇类芳香物质E-香叶醇的生物合成。 3.利用基因组关联性分析(GWAS)分析，发现了脂肪酸途径醇类芳香物质合成相关的2个候选基因。开展了398份番茄果实材料的基因组重测序，以及芳香物质含量分析，基于GWAS分析技术获得了同脂肪酸途径的己醇和Z-3-己烯醇等醇类芳香物质形成相关的SNPs位点。进一步分析发现在其上下游0.5 Mb区域内存在候选基因，其中Solyc09g091050是一种脂肪酶编码基因，为开展脂肪酸途径醇类芳香物质的基因功能鉴别与功能验证提供了依据。 4.根据基因编辑的番茄果实表型，鉴别出了调控脂肪酸途径醇类芳香物质合成的基因。利用CRISPR／cas9基因编辑技术敲除了脂肪酶(Lipase)编码基因Solyc09g091050，结果显示基因片段中发生了5 bp碱基的删减，由此导致了脂肪酶移码突变。表型鉴定后发现与野生型番茄果实相比，突变果实的己醇，Z-3-己烯醇，1-戊醇等醇类芳香物质含量显著减少。利用重组蛋白开展体外酶活性分析，结果显示脂肪酶可催化甘油三亚油酸酯形成醇类芳香物质生物合成的前体物质亚油酸，表明脂肪酶是脂肪酸途径上重要的初始酶。 关键词：AP2-ERF；芳香物质；基因编辑；基因组关联性分析；萜类合成酶；脂肪酶

 Aromatic alcohols are important volatile components of fruit quality, and could be classified into different chemical classes according to structures, including linear chains, terpenoids and benzene-containing. Aromatic alcohols can be synthesized by different pathways using terpenoid and fatty acids as substrates. Different fruits have different characteristic aromatic alcohol components. For example, E-geraniol derived from the terpenoid pathway contribute to citrus fruit aroma, and C5 and C6 alcohols from fatty acid pathway are important to tomato fruit aroma. In this study, citrus and tomato fruits were used as materials to identify genes associated to aromatic alcohol biosynthesis, and then to identify transcription factors involved in volatile alcohol formation. Main results were as follows: 1.Based on in vitro expression and homologous transient overexpression assay, CitTPS16 was involved in E-geraniol biosynthesis. Using the sweet orange genome database, the terpenoid synthase (TPS) gene was cloned, 10 TPS recombinant proteins were constructed, and the enzymatic activity was analyzed. It was found that CitTPS16 belonging to the TPS-b subfamily was able to catalyze in vitro formation of E-geraniol using geranyl diphosphate (GPP). Hereby, transient overexpression analysis of CitTPS16 was performed in citrus leaves and fruits, respectively, and it was found that overexpression of CitTPS16 significantly promoted the increase of E-geraniol content. These results indicated that CitTPS16 is associated with the biosynthesis of E-geraniol, which provided a target gene for transcriptional regulation research. 2.Based on the dual-luciferase, yeast-one hybrid and EMSA assays, the transcription factor CitERF71, which was able to trans-activate structural gene CUTPS16, was identified. Using the sweet orange genome database, 48 AP2/ERF family members were cloned. The results of tobacco dual luciferase assays were shown that CitERF71 is capable to trans-activate the promoter activity of CitTPS16. During the development of citrus fruit, the expression of CUERF71 was positively correlated with CitTPS16 expression (r = 0.79, P<0.05) and E-geraniol content (r = 0.96, P<0.05). Yeast-one hybrid and EMSA assay demonstrated specificity of binding CitERF71 to the ACCCGCC and GGCGGG sequences of the CitTPS16 promoter. These comprehensive results showed that CitERF71 is involved in transcriptional regulation of CitTPS16, and therefore, in controlling production of is-geraniol in citrus fruit. 3.Using genomic association analysis study (GWAS), two candidate genes related to the synthesis of aromatic alcohols in fatty acid pathway were discovered. Genomic resequencing of 398 tomato fruit materials and analysis of aromatic content were carried out. Based on GWAS analysis technique, SNPs related to the formation of aromatic alcohols, such as hexanol and Z-3-hexenol, were obtained. Further analysis revealed that there are candidate genes in the upstream and downstream of 0.5 Mb region, among which, Solyc09g091050 is a lipase-encoding gene which provides a basis for the functional identification and functional verification of aromatic alcohols in fatty acid pathway. 4.Based on the phenotype of the genetically edited tomato fruits, genes that regulate the synthesis of aromatic alcohols in the fatty acid pathway were identified. Using the CRISPR/cas9 gene editing technique to knock out the lipase-encoding gene Solyc09g091050, the result showed that a 5 bp base deletion occurred in the gene coding region, resulting in a lipase frameshift mutation. After phenotypic identification, it was found that the content of aromatic alcohols, such as hexanol, Z-3-hexenol and 1-pentanol of the mutant fruits, were significantly reduced, compared with the wild type tomato fruits. In vitro enzymatic activity analysis using recombinant protein showed that lipase was able to catalyze the formation of linoleic acid, a precursor of biosynthesis of aromatic alcohols, which indicates that lipase is an important initial enzyme in the fatty acid pathway. Key words: AP2-ERF; CRISPR-cas9; GWAS; LIPASE; TPS; volatiles