本文選題：煙草 + 釀酒酵母��； 參考：《中國農(nóng)業(yè)科學院》2016年博士論文

【摘要】：長鏈多不飽和脂肪酸(long chain polyunsaturated fatty acids,LC-PUFAs)對人體有良好生理功能,除了魚油這種傳統(tǒng)來源外,在油料作物中重構(gòu)LC-PUFAs的合成途徑是一種非常有前景的替代來源。在以表達脂連接型去飽和酶為策略的LC-PUFAs代謝工程研究中,去飽和酶中間代謝產(chǎn)物需要在PC庫和CoA庫之間進行多次轉(zhuǎn)換,導致LC-PUFAs合成的終產(chǎn)物含量較低。LPCAT(屬于溶血磷脂�；D(zhuǎn)移酶)被推測是在轉(zhuǎn)換過程中起重要作用的�；D(zhuǎn)移酶。然而,植物來源的LPCAT報道較少,而且LPCAT對LC-PUFAs合成的影響以及參與PC和CoA庫之間脂肪�；D(zhuǎn)運的機制尚不清楚。本研究從煙草Nicotiana benthamiana和三角褐指藻Phaeodactylum tricornutum中克隆LPCAT基因,并在酵母中進行功能驗證和底物特異性研究。通過將LPCAT與不同類型脂肪酸去飽和酶和延長酶在酵母中共表達,分析其脂肪酸,CoA及脂質(zhì)組變化,試圖闡明LPCAT對LC-PUFAs合成的中間代謝產(chǎn)物轉(zhuǎn)運及終產(chǎn)物積累的影響機制。主要結(jié)果如下:(1)從煙草中克隆到兩個溶血磷脂�；D(zhuǎn)移酶基因NbLPCAT1和NbLCPAT2,其編碼產(chǎn)物均屬于MBOAT蛋白家族。LysoPAF敏感實驗表明NbLPCAT1和NbLCPAT2都有LPCAT酰基轉(zhuǎn)移酶活性。酵母磷脂分析實驗表明:表達NbLPCAT1和NbLPCAT2的重組酵母優(yōu)先利用16:0-LPC,16:1-LPC和18:1-LPC作為�；荏w;18:2-CoA,18:3-CoA為�；w。利用重組酵母微粒體為LPCAT酶源的體外酶活實驗表明:NbLPCAT1對LPC及不飽和的C18-CoAs表現(xiàn)出偏好性。NbLPCAT2對LPA表現(xiàn)出的活性比LPC要高,且對18:3-CoA有較好的偏好性。采用RT-PCR方法檢測煙草LPCAT基因的表達模式,結(jié)果表明:NbLPCAT1和NbLPCAT2在花中的表達水平最高,在其它組織器官中均有不同程度的表達。(2)從富含EPA的三角褐指藻中克隆到一個PtLPCAT基因并驗證功能。將NbLPCAT1,NbLPCAT2及PtLPCAT與不同來源的脂肪酸去飽和酶和延長酶在酵母中共表達,這些脂肪酸去飽和酶和延長酶包括:來源于三角褐指藻的脂連接型去飽和酶PtD6,PtD5;來源于小立碗蘚的脂肪酸延長酶PSE;來源于青綠藻的CoA型去飽和酶OtD6和細小微胞藻中的CoA型去飽和酶MsD5。構(gòu)建三種不同類型重組酵母并進行功能驗證,三種類型的重組酵母中分別共表達:(a)CoA型去飽和酶和延長酶;(b)脂連接型去飽和酶和延長酶;(c)脂連接型去飽和酶、延長酶和LPCAT。結(jié)果表明:(a)類型重組酵母中LC-PUFAs終產(chǎn)物的含量最高,(c)類型重組酵母與(b)類型重組酵母相比,△6去飽和酶中間代謝產(chǎn)物在CoA庫中的含量較高,終產(chǎn)物LC-PUFAs的積累量較高,表明LPCAT的表達促進了代謝中間產(chǎn)物的脂肪�；赑C和CoA之間的轉(zhuǎn)移,在一定程度上解除脂連接型去飽和酶應用于轉(zhuǎn)基因合成LC-PUFAs過程中的代謝瓶頸,提高LC-PUFAs的積累量。(3)不同類型重組酵母脂質(zhì)組分析表明:(c)類型重組酵母與(b)類型重組酵母相比,表達NbLPCAT1的重組酵母菌株中PCsn-1、sn-2位置上18:2和18:3脂�；暮棵黠@上調(diào),表達NbLPCAT2的重組酵母菌株中PCsn-1位置18:2脂�；可险{(diào)。表明LPCAT的表達能夠明顯提高△6去飽和酶反應的底物和產(chǎn)物的量;同時進一步證實了NbLPCAT1和NbLPCAT2的底物偏好性,也表明了NbLPCAT1對LPC�；奈恢脹]有明顯的偏好性。
[Abstract]:Long chain polyunsaturated fatty acids (long chain polyunsaturated fatty acids, LC-PUFAs) have good physiological functions for human body. Apart from the traditional source of fish oil, the synthesis of LC-PUFAs in oil crops is a very promising alternative source. In the study of LC-PUFAs metabolic engineering with the strategy of expressing fat linked desaturase as a strategy. In the study, the intermediate metabolites of the desaturase need to undergo multiple transformations between the PC library and the CoA library, resulting in the lower.LPCAT (lysophosphatidic acyl transferase) of the LC-PUFAs synthesis (lysophosphatidyl transferase) is presumed to be an acyl transferase that plays an important role in the conversion process. However, the LPCAT reports from the source of the plant are less, and LPCAT is compatible with LC-PUFAs. The mechanism of fatty acyl transport between PC and CoA libraries is not clear. This study cloned the LPCAT gene from tobacco Nicotiana benthamiana and Phaeodactylum tricornutum of brown finger alga, and carried out functional verification and substrate specificity in yeast. By depressing LPCAT and different types of fatty acids to desaturase and prolonging it. Long enzyme was expressed in yeast, and the changes of fatty acids, CoA and lipid groups were analyzed. The effects of LPCAT on the transport of intermediate metabolites and the accumulation of final products in the synthesis of LC-PUFAs were elucidated. The main results were as follows: (1) two lysophosphatidic acyl transferase based on NbLPCAT1 and NbLCPAT2 were cloned from tobacco. All the encoding products were MBOAT eggs. The.LysoPAF sensitive experiment in the white family showed that both NbLPCAT1 and NbLCPAT2 had LPCAT acyl transferase activity. The yeast phospholipid analysis experiment showed that the recombinant yeast expressing NbLPCAT1 and NbLPCAT2 preferred 16:0-LPC, 16:1-LPC and 18:1-LPC as acyl receptor, 18:2-CoA, 18:3-CoA as acyl donor. The recombinant yeast microsome was the body of the LPCAT enzyme source. The experiment of external enzyme activity showed that NbLPCAT1 showed higher activity to LPA than LPC in LPC and unsaturated C18-CoAs, and had better preference to 18:3-CoA. RT-PCR method was used to detect the expression pattern of LPCAT gene in tobacco. The results showed that the expression level of NbLPCAT1 and NbLPCAT2 in the flowers was the highest, in other tissues. There were different levels of expression. (2) a PtLPCAT gene was cloned from EPA rich brown finger alga and the function was verified. NbLPCAT1, NbLPCAT2 and PtLPCAT were expressed in yeast with different sources of fatty acid desaturase and elongate enzyme. These fatty acid desaturase and elongate enzymes include lipid connections derived from brown finger alga Type desaturase PtD6, PtD5, fatty acid elongate enzyme PSE derived from the small bowl moss, CoA dehydrogenase OtD6 from green algae and CoA dehydrogenase MsD5. in microcya, and construct three different types of recombinant yeast and perform functional verification. The three types of recombinant yeast are co expressed as (a) CoA type desaturase and lengthening enzyme; (b) fat linked desaturase and extended enzyme; (c) fat linked desaturase, extended enzyme and LPCAT. results showed that the content of LC-PUFAs end products in (a) type of recombinant yeast was the highest, and (c) type recombinant yeast compared with (b) type of recombinant yeast, the content of delta 6 desaturase intermediate metabolites in CoA library was higher, and the accumulation of final product LC-PUFAs It is suggested that the expression of LPCAT promotes the transfer of fatty acyl groups between PC and CoA, and to some extent relieves the metabolic bottleneck of the use of fat linked desaturase in the process of transgene synthesis of LC-PUFAs and increases the accumulation of LC-PUFAs. (3) the analysis of different types of recombinant yeast lipid groups showed: (c) type of recombinant yeast Compared with (b) type of recombinant yeast, the content of 18:2 and 18:3 lipoyl groups in the recombinant yeast strains expressing NbLPCAT1 was up obviously up, and the 18:2 acyl content of PCsn-1 position in the recombinant yeast expressing NbLPCAT2 was up to be up, indicating that the expression of LPCAT could obviously improve the amount of the substrate and product of the reaction of delta 6 desaturase. It further confirmed the substrate preference of NbLPCAT1 and NbLPCAT2, and also showed that NbLPCAT1 had no obvious preference for LPC acylation sites.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q943.2

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