枸杞中二咖啡酰多胺类化合物生物合成途径关键酶SHT基因克隆及功能研究

Dicaffeoylpolyamines derivatives, the main active ingredients of the wolfberry, have a variety of pharmacological effects and great economic value. However, it is difficult to separate these kind of composition, and the metabolic engineering can regulates its biosynthesis to increase the yield. Spermidine or spermine hydroxycinnamoyl transferase（SHT） is the key enzyme in dicaffeoylpolyamines derivatives biosynthesis and an important target in metabolic engineering, but it has not yet been characterized. After metabolomic and transcriptomic comparison of the dicaffeoylpolyamines derivatives wild and cultivated wolfberry, we will partially purify the crude enzyme of 4-coumaryl:CoA ligase and prepared donator for SHT. Based on the above results, we are going to select the predicted SHT genes and construct escherichia coli engineering strains to verify the function of predicted genes through enzymatic reaction in vitro.We will also about to investigate the content distribution of dicaffeoylpolyamines derivatives in wild and cultivated wolfberry.In this study, we intended to clone and analyze the function of SHT gene, which encode the key enzyme in dicaffeoylpolyamines derivatives biosynthesis of wolfberry, through comprehensive bioinformatics, chemistry, and molecular biology methods. The expected outcomes will provide a new target for the regulation of dicaffeoylpolyamines derivatives biosynthesis and pave the way for the clarification of the further pathway, which may help to understand the molecular formation mechanism between wild and cultured wolfberry.

野生枸杞含有二咖啡酰精胺类化合物，栽培枸杞多含有二咖啡酰亚精胺类化合物，两种二咖啡酰多胺类化合物是枸杞果实中一类特征性化合物，具有显著药理活性和经济价值。但其为两性化合物，分离难度大，代谢工程调控其生物合成可提高产量。多胺羟基肉桂酰基转移酶（SHT）是二咖啡酰多胺类物质生物合成途径关键酶和重要调控靶标，迄今国内外尚未克隆鉴定。本课题分析野生枸杞和栽培枸杞之间代谢组和转录组差异后，制备了羟基肉桂酰-CoA。据此克隆SHT基因，在大肠杆菌外源表达验证功能。并考察二咖啡酰多胺类化合物在野生枸杞与栽培枸杞中分布情况。本课题综合运用生物信息学，化学和分子生物学方法挖掘SHT基因并研究二咖啡酰多胺在枸杞体内的分布情况，为利用代谢工程手段调控二咖啡酰多胺类化合物生物合成提供全新靶标，为解析后继生物合成途径奠定基础，并初步阐述了野生枸杞与栽培枸杞中二咖啡酰多胺类成分差异的分子机制。

枸杞中的二咖啡酰多胺类化合物是一类重要的酚酰胺类化合物，近年来由于其独特构型和可能的活性而备受关注，吸引了越来越多研究人员的关注。但是对其合成途径上酰基转移酶功能的研究却尚未开展。本项目以二咖啡酰多胺类成分合成途径上关键的酰基转移酶为研究目标，运用转录组测序，基因全长克隆及基因体外功能验证等方式对枸杞的酰基转移酶进行研究。同时以前期研究中获得的二咖啡酰多胺类化合物为标准品，对不同来源和不同发育阶段的枸杞果实中此类物质进行定量分析。取得的研究成果如下：1.初步阐述了枸杞果实中二咖啡酰多胺类化合物的分布规律；2.获得了一个全面的枸杞转录组数据；3.筛选并克隆鉴定了12条酰基转移酶基因，通过体外功能研究获得了催化生成咖啡酰多胺类的酶。研究结果首次较为系统地阐述了枸杞果实中二咖啡酰多胺类物质的分布规律，并对其合成的酰基转移酶进行了初步的功能研究，本研究为此类物质的合成生物学研究奠定了基础。在本项目的资助下，以第一作者或通讯作者发表学术论文2篇，其中SCI收录1篇，国内核心期刊1篇，培养硕士研究生1名。