發(fā)布時(shí)間：2018-04-27 23:05

  本文選題：蠟梅 + 花香　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：蠟梅(ChimonanthuspraecoxL.)為我國傳統(tǒng)名花,是蠟梅科蠟梅屬落葉叢生灌木�；ㄏ沭ビ�,開于百花寂寥的冬季,香氣特征作為重要性狀,在切花、園林種植中廣泛應(yīng)用。本文以芳香的H29和無香的SW001品系蠟梅作為研究材料,測定4個(gè)時(shí)期揮發(fā)物成分。篩選轉(zhuǎn)錄組庫萜類差異表達(dá)基因CpTPS5、CpTPS16,通過實(shí)時(shí)定量表達(dá)分析,結(jié)合揮發(fā)物種類及釋放量差異,尋找基因表達(dá)與花香釋放之間的聯(lián)系。構(gòu)建雙元表達(dá)載體,在煙草中超量表達(dá)CpTPS5、CpTPS16,利用GC-MS對(duì)香氣成分定性及定量分析,初步探究萜烯合成酶功能。1、H29和SW001蠟梅品系共鑒定出64種香氣成分,萜類、苯環(huán)型化合物占據(jù)主導(dǎo)地位。H29盛花期單位時(shí)間內(nèi)揮發(fā)物質(zhì)總量為3449.48ng/g,含量較高的物質(zhì)分別為:芳樟醇、乙酸芐酯、水楊酸甲酯。SW001同期揮發(fā)物總量為9886.26ng/g,含量較高的物質(zhì)分別為:反式-β羅勒烯、(1S,2S,3R,5S)-(+)-2,3-蒎烷二醇、2,6-二甲基-1,3,5,7-辛四烯、甜樺油、苯甲酸甲酯。2、對(duì)2品系4個(gè)時(shí)期花香成分測定,H29萜類總含量露瓣期至末花期,逐漸增加。單萜含量變化一致;倍半萜最高含量86.57ng/g出現(xiàn)于露瓣期,隨后降低,末花期有所回升。SW001萜類總含量變化趨勢(shì)呈先增后減,單萜揮發(fā)物釋放與之一致;倍半萜揮發(fā)物含量露瓣期高于初開期,于盛花期達(dá)到頂峰,為44.46ng/g,末花期降低。3、經(jīng)蛋白序列比對(duì),CpTPS5、CpTPS16在H29和SW0012品系中無差異;構(gòu)建系統(tǒng)進(jìn)化樹,CpTPS5與TPSa倍半萜合酶家族聚類到一起,與石竹烯較為相近;CpTPS16同TPSb單萜合酶萜家族聚類到一起。4、實(shí)時(shí)定量PCR結(jié)果顯示:CpTPS5基因在H29中上調(diào)表達(dá),表達(dá)先增加后減少,盛花期表達(dá)量是同期SW001品系13倍;CpTPS16基因在SW001中上調(diào)表達(dá),在4時(shí)期中顯著高于基因在H29品系表達(dá)量。2品系中表達(dá)趨勢(shì)相同,由露瓣期至末花期持續(xù)增加。5、煙草中超量表達(dá)CpTPS5、CpTPS16,由揮發(fā)物成分測定及定量計(jì)算分析可得:萜類成分中單萜、倍半萜物質(zhì)釋放量呈現(xiàn)規(guī)律性,轉(zhuǎn)基因植株與野生型揮發(fā)物種類無顯著差異,但揮發(fā)性萜類遠(yuǎn)超于野生型中萜類釋放量。絕對(duì)定量計(jì)算石竹烯、芳樟醇、檸檬烯、萘?、己酸甲酯5種揮發(fā)性物質(zhì),發(fā)現(xiàn)在轉(zhuǎn)CpTPS16基因中上述萜類物質(zhì)釋放量約為CpTPS5中2倍,且顯著高于其在野生型、空載中的釋放量,而鏈狀酯類己酸甲酯在CpTPS5、CpTPS16釋放量基本持平,無明顯差異。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S685.99

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本文編號(hào)：1812730