【作者】 陈杉艳；

【导师】 伊华林；

【作者基本信息】 华中农业大学 ， 果树学， 2011， 硕士

【摘要】 近年来,对柑橘果实香气物质的研究主要集中在发现新的香气物质和建立新的检测方法等方面。尽管在柑橘中已鉴定出200多种香气物质,但有关这些物质的生物合成途径及其调控机制的研究报道却很少。为了探索柑橘果实香气物质的生物合成与其关键基因之间的关系,本论文以红肉脐橙(Citrus sinensis Osbeck cv. Cara Cara)和华盛顿脐橙(Citrus sinensis Osbeck cv. Washington)果实为研究对象,采用固相微萃取(SPME)和气相色谱、质谱联用(GC-MS)技术对实验材料的香气成分进行了分析,克隆了柑橘果实香气代谢过程中的3个关键基因(牻牛儿基焦磷酸合成酶基因Csgpps、d-柠檬烯合成酶基因Csdlims和芳樟醇合成酶基因Cslinas),并通过实时荧光定量PCR技术(RT-qPCR)对这三个基因做了表达分析。主要研究结果如下：1.以环己酮为内标,d-柠檬烯为标样,利用SPME-GC-MS定量法测定了红肉脐橙和华盛顿脐橙不同成熟期果皮和果肉中d-柠檬烯的含量。结果表明：红肉脐橙果皮d-柠檬烯呈现先降低后升高最后再降低的变化趋势；果肉d-柠檬烯在早期变化不大,后期含量迅速升高。华盛顿脐橙果皮和果肉d-柠檬烯则呈现出几乎一致的变化趋势,即先升高后再降低的变化趋势。这表明不同的柑橘品种其香气物质的变化规律不同,相同品种不同组织中香气物质呈现出相同或不同的变化规律。2.以环己酮为内标,利用SPME-GC-MS半定量法测定了红肉脐橙和华盛顿脐橙不同成熟期果皮和果肉中主要香气物质的含量变化。结果表明：红肉脐橙果皮中的主要香气物质α-蒎烯、β-月桂烯、γ-萜品烯在果实成熟过程中的变化趋势相似,即在花后210d时含量达最高值,β-芳樟醇的变化趋势则与上述3种物质相反。红肉脐橙果肉中的主要香气物质α-蒎烯、β-月桂烯、γ-萜品烯、巴伦西亚橘烯在果实成熟发育过程中也呈现相似的变化趋势,在花后220d时含量均迅速降低为零,果肉中没有检测到β-芳樟醇这种物质。华盛顿脐橙果皮中α-蒎烯、β-月桂烯和γ-萜品烯均呈现直线升高的变化趋势,β-芳樟醇则在花后200d到210d期间略有下降。果肉中α-蒎烯、β-月桂烯和γ-萜品烯在花后190d到200d期间均呈现迅速降低后保持不变的趋势,巴伦西亚橘烯的变化趋势则与上述3种物质不同,果肉中也没有检测到β-芳樟醇这种物质。3.利用RT-qPCR技术对克隆到的关键酶基因进行了表达分析。结果发现红肉脐橙和华盛顿脐橙果肉成熟过程中Csdlims基因的上调表达和下调表达的变化趋势与果肉中d-柠檬烯含量的变化趋势一致,Cslinas基因在两种脐橙果肉中表达量很低(表达量几乎为零),这与果肉中检测不到β-芳樟醇这种香气物质相符合,由此说明,柑橘果实中主要香气物质的含量变化与其关键基因的表达具有密切联系。更多还原

【Abstract】 In recent years, the investigation on aroma of citrus fruits is mainly concentrated on discovering novel volatiles and establishing new qualitative or quantitative determination methods. In spite of that more than 200 kinds of volatiles have been identified in citrus, the biosynthetic pathway and its regulatory mechanism have rarely been studied.In this study, we attempted to explore the correlation between the biosynthesis of citrus aroma compounds and its related key genes. Cara Cara navel orange(Citrus sinensis Osbeck cv. Cara Cara) and Washington navel orange(Gitrus sinensis Osbeck cv. Washington) were used as experimental materials. Aroma compounds of these fruits were extracted by head-space solid-phase microextraction and determined by Gas Chromatogram/Mass Spectrum. Three key genes (Csgpps. Csdlims and Cslinas) in the biosynthetic pathway have been cloned. Furthermore, the expression of these genes during citrus fruits maturation was analyzed by real-time quantity PCR. The main results show as follows:1. The content of d-limonene from citrus flavedo and pulp was determined by SPME-GC-MS, which used cyclohexanone as internal standard as well as d-limonene as external standard. The content of d-limonene from Cara Cara navel orange flavedo displayed a tendency that decreased at the early mature stage and raised gradually at the middle mature stage, then dropped again at the last stage. While the content of d-limonene from its pulp maintained a stable level at the early mature stage, and then soared at the last stage. As for the Washington navel orange, the content of d-limonene from its flavedo and pulp shared an almost identical trend, which increased at the early mature stage and then decreased. This indicated that the aroma in different citrus varieties presented diverse variation patten. While the aroma in different tissues from the same variety shared the similar or distinct variation patten. 2. Contents of the main volatiles from Cara Cara and Washington navel orange flavedo and pulp during different stages of maturity were determined using semi-quantitative method by SPME-GC-MS, which utilized cyclohexanone as the internal standard. The main volatiles a-pinene,β-myrcene, r-terpinene from Cara Cara navel orange flavedo had the similar variation patten during fruits maturation, and all of them reached a maximum content at 210 days after flowering. However, the variation patten of B-linalool were contrary to the above three kinds of volatiles. a-pinene,β-myrcene, r-terpinene and valencene from Cara Cara navel orange pulp also showed the similar trends, which decreased rapidly to zero at 220 days after flowering.α-pinene, B-myrcene, and r-terpinene from Washington navel orange flavedo presented a linear increase during fruits maturation, whileβ-linalool declined slightly during 200 days to 210 days after flowering.α-pinene, B-myrcene and r-terpinene from Washington navel orange pulp decreased suddenly during 190 days to 200 days after flowering and then remained unchanged, while valencene showed a different variation patten from the above three kinds of aromas. In addition,β-linalool was not detected in neither Cara Cara nor Washington navel orange pulp.3. Expression of key genes for volatile biosyntheis in citrus was analyzed by real-time quantitative PCR. The results declared that the expression variation pattern of Csdlims gene was in accordance with the variation patten of its product during fruit maturation both in Cara Cara and Washington navel orange pulp. The expression of Cslinas gene was very low in the above two citrus varieties, which could be regarded as nearly no expression. Similarly, the product of Cslinas gene was not determined in both of the above citrus. This implied that the content of main volatiles in citrus was closely connected with the expression of its key genes.更多还原