蜡梅花香挥发物测定及2个萜烯合酶基因功能初步研究

发布时间：2018-04-27 23:05

本文选题：蜡梅 + 花香　；参考：《华中农业大学》2017年硕士论文

【摘要】：蜡梅(ChimonanthuspraecoxL.)为我国传统名花,是蜡梅科蜡梅属落叶丛生灌木。花香馥郁,开于百花寂寥的冬季,香气特征作为重要性状,在切花、园林种植中广泛应用。本文以芳香的H29和无香的SW001品系蜡梅作为研究材料,测定4个时期挥发物成分。筛选转录组库萜类差异表达基因CpTPS5、CpTPS16,通过实时定量表达分析,结合挥发物种类及释放量差异,寻找基因表达与花香释放之间的联系。构建双元表达载体,在烟草中超量表达CpTPS5、CpTPS16,利用GC-MS对香气成分定性及定量分析,初步探究萜烯合成酶功能。1、H29和SW001蜡梅品系共鉴定出64种香气成分,萜类、苯环型化合物占据主导地位。H29盛花期单位时间内挥发物质总量为3449.48ng/g,含量较高的物质分别为:芳樟醇、乙酸苄酯、水杨酸甲酯。SW001同期挥发物总量为9886.26ng/g,含量较高的物质分别为:反式-β罗勒烯、(1S,2S,3R,5S)-(+)-2,3-蒎烷二醇、2,6-二甲基-1,3,5,7-辛四烯、甜桦油、苯甲酸甲酯。2、对2品系4个时期花香成分测定,H29萜类总含量露瓣期至末花期,逐渐增加。单萜含量变化一致;倍半萜最高含量86.57ng/g出现于露瓣期,随后降低,末花期有所回升。SW001萜类总含量变化趋势呈先增后减,单萜挥发物释放与之一致;倍半萜挥发物含量露瓣期高于初开期,于盛花期达到顶峰,为44.46ng/g,末花期降低。3、经蛋白序列比对,CpTPS5、CpTPS16在H29和SW0012品系中无差异;构建系统进化树,CpTPS5与TPSa倍半萜合酶家族聚类到一起,与石竹烯较为相近;CpTPS16同TPSb单萜合酶萜家族聚类到一起。4、实时定量PCR结果显示:CpTPS5基因在H29中上调表达,表达先增加后减少,盛花期表达量是同期SW001品系13倍;CpTPS16基因在SW001中上调表达,在4时期中显著高于基因在H29品系表达量。2品系中表达趋势相同,由露瓣期至末花期持续增加。5、烟草中超量表达CpTPS5、CpTPS16,由挥发物成分测定及定量计算分析可得:萜类成分中单萜、倍半萜物质释放量呈现规律性,转基因植株与野生型挥发物种类无显著差异,但挥发性萜类远超于野生型中萜类释放量。绝对定量计算石竹烯、芳樟醇、柠檬烯、萘?、己酸甲酯5种挥发性物质,发现在转CpTPS16基因中上述萜类物质释放量约为CpTPS5中2倍,且显著高于其在野生型、空载中的释放量,而链状酯类己酸甲酯在CpTPS5、CpTPS16释放量基本持平,无明显差异。
[Abstract]:Chimonanthus praecox L. It is a traditional famous flower in China, which is a deciduous bush. Fragrant flowers bloom in the lonely winter, aroma characteristics as an important trait, widely used in cut flowers, garden planting. In this paper, aromatic H29 and unscented SW001 were used as the research materials to determine the volatile components in four periods. Terpenoid differential expression gene CpTPS5 (CpTPS5) CpTPS16 was screened, and the relationship between gene expression and flower aroma release was explored by real-time quantitative expression analysis, combined with the difference of volatile species and release amount. A double component expression vector was constructed to overexpress CpTPS5CpTPS16 in tobacco. The quantitative and qualitative analysis of aroma components was carried out by GC-MS, and 64 aroma components, terpenes, were identified by using the function of terpene synthase .1H29 and SW001 waxy plum lines. The total amount of volatile matter per unit time in flowering stage of benzene ring compound is 3449.48 ng / g, and the higher content is linalool, benzyl acetate, benzyl acetate. The total volatiles of methyl salicylate, SW001, were 9886.26ng / g at the same time, and the higher contents were as follows: trans- 尾 -rotene, 1SN ~ 2S ~ (2 +) S ~ (3 +) R ~ (5) S ~ (-) ~ (5) S ~ (-) ~ (2) ~ (2) -pinane diol ~ (2-) ~ (2) -dimethyl-357 ~ (-octylenes), and Betula platyphylum oil, Methyl benzoate. 2. The total content of H29 terpenoids was determined in 4 periods of 2 strains and increased gradually from the period of dew to the end of flowering. The maximum content of sesquiterpene 86.57ng/g appeared in the open valve stage and then decreased. The total content of SW001 terpenoids increased first and then decreased, and the release of monoterpene volatiles was consistent with the change of monoterpene content. The content of sesquiterpene volatiles was higher in open valve stage than in early flowering stage, and reached its peak at full flowering stage, which was 44.46 ng / g, and decreased by .3. the protein sequence of CpTPS5CpTPS16 was not different between H29 and SW0012 strains, and the phylogenetic tree CpTPS5 was clustered with TPSa sesquiterpene synthase family. Close to caryophyllene, CpTPS16 was clustered with TPSb monoterpene synthase family. The results of real-time quantitative PCR showed that the expression of CpTPS5 gene was up-regulated in H29, and the expression increased first and then decreased. The expression level of CpTPS16 gene in SW001 was 13 times higher than that in H29 strain at 4 stages, and the expression trend of CpTPS16 gene in H29 strain was the same as that in H29 strain at 4 stages, and the expression level of CpTPS16 gene in H29 strain was significantly higher than that in H29 line. The expression of CpTPS5 and CpTPS16 in tobacco increased continuously from the open valve stage to the late flowering stage. The monoterpenes and sesquiterpenes released from the terpenoids were obtained by quantitative analysis and determination of the volatile components, and the release of sesquiterpenes was regular. There was no significant difference between transgenic plants and wild type volatile species, but the amount of volatile terpenoids was much higher than that of wild type. Five volatile substances, including caryophyllene, linalool, limonene, naphthalene and methyl caproate, were calculated quantitatively. It was found that the release of these terpenoids in the transgenic CpTPS16 gene was about 2 times of that in CpTPS5, and significantly higher than that in wild type and no load. However, the release of chain ester methyl caproate at CpTPS5 and CpTPS16 was almost the same, but there was no significant difference.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S685.99

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