溶血磷脂酰基转移酶的鉴定及其对转基因合成长链多不饱和脂肪酸的影响研究

发布时间：2018-07-08 19:26

  本文选题：烟草 + 酿酒酵母　； 参考：《中国农业科学院》2016年博士论文

【摘要】：长链多不饱和脂肪酸(long chain polyunsaturated fatty acids,LC-PUFAs)对人体有良好生理功能,除了鱼油这种传统来源外,在油料作物中重构LC-PUFAs的合成途径是一种非常有前景的替代来源。在以表达脂连接型去饱和酶为策略的LC-PUFAs代谢工程研究中,去饱和酶中间代谢产物需要在PC库和CoA库之间进行多次转换,导致LC-PUFAs合成的终产物含量较低。LPCAT(属于溶血磷脂酰基转移酶)被推测是在转换过程中起重要作用的酰基转移酶。然而,植物来源的LPCAT报道较少,而且LPCAT对LC-PUFAs合成的影响以及参与PC和CoA库之间脂肪酰基转运的机制尚不清楚。本研究从烟草Nicotiana benthamiana和三角褐指藻Phaeodactylum tricornutum中克隆LPCAT基因,并在酵母中进行功能验证和底物特异性研究。通过将LPCAT与不同类型脂肪酸去饱和酶和延长酶在酵母中共表达,分析其脂肪酸,CoA及脂质组变化,试图阐明LPCAT对LC-PUFAs合成的中间代谢产物转运及终产物积累的影响机制。主要结果如下:(1)从烟草中克隆到两个溶血磷脂酰基转移酶基因NbLPCAT1和NbLCPAT2,其编码产物均属于MBOAT蛋白家族。LysoPAF敏感实验表明NbLPCAT1和NbLCPAT2都有LPCAT酰基转移酶活性。酵母磷脂分析实验表明:表达NbLPCAT1和NbLPCAT2的重组酵母优先利用16:0-LPC,16:1-LPC和18:1-LPC作为酰基受体;18:2-CoA,18:3-CoA为酰基供体。利用重组酵母微粒体为LPCAT酶源的体外酶活实验表明:NbLPCAT1对LPC及不饱和的C18-CoAs表现出偏好性。NbLPCAT2对LPA表现出的活性比LPC要高,且对18:3-CoA有较好的偏好性。采用RT-PCR方法检测烟草LPCAT基因的表达模式,结果表明:NbLPCAT1和NbLPCAT2在花中的表达水平最高,在其它组织器官中均有不同程度的表达。(2)从富含EPA的三角褐指藻中克隆到一个PtLPCAT基因并验证功能。将NbLPCAT1,NbLPCAT2及PtLPCAT与不同来源的脂肪酸去饱和酶和延长酶在酵母中共表达,这些脂肪酸去饱和酶和延长酶包括:来源于三角褐指藻的脂连接型去饱和酶PtD6,PtD5;来源于小立碗藓的脂肪酸延长酶PSE;来源于青绿藻的CoA型去饱和酶OtD6和细小微胞藻中的CoA型去饱和酶MsD5。构建三种不同类型重组酵母并进行功能验证,三种类型的重组酵母中分别共表达:(a)CoA型去饱和酶和延长酶;(b)脂连接型去饱和酶和延长酶;(c)脂连接型去饱和酶、延长酶和LPCAT。结果表明:(a)类型重组酵母中LC-PUFAs终产物的含量最高,(c)类型重组酵母与(b)类型重组酵母相比,△6去饱和酶中间代谢产物在CoA库中的含量较高,终产物LC-PUFAs的积累量较高,表明LPCAT的表达促进了代谢中间产物的脂肪酰基在PC和CoA之间的转移,在一定程度上解除脂连接型去饱和酶应用于转基因合成LC-PUFAs过程中的代谢瓶颈,提高LC-PUFAs的积累量。(3)不同类型重组酵母脂质组分析表明:(c)类型重组酵母与(b)类型重组酵母相比,表达NbLPCAT1的重组酵母菌株中PCsn-1、sn-2位置上18:2和18:3脂酰基的含量明显上调,表达NbLPCAT2的重组酵母菌株中PCsn-1位置18:2脂酰基含量上调。表明LPCAT的表达能够明显提高△6去饱和酶反应的底物和产物的量;同时进一步证实了NbLPCAT1和NbLPCAT2的底物偏好性,也表明了NbLPCAT1对LPC酰基化的位置没有明显的偏好性。
[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.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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