宁夏枸杞玉米黄素双棕榈酸酯酰基转移酶基因功能解析

The dried fruits of Lycium barbarum L., commonly known as Goji, being rich in bioactive compounds such as esterified carotenoids potentially serving to "Yang Gan Min Mu", become popular health care products. It was reported so far that the content of zeaxanthin dipalmitate (ZDP) in Goji is the highest than other plant tissues. Although the biosynthetic route of carotenoid in model species is relatively clear, the universal and conservative molecular mechanism underlying of carotenoid esterification promoting its biosynthesis and storage in fruits and vegetables remains unclear. An integrative approach was utilized to predict the candidate gene, zeaxanthin dipalmitate acyltransferase (LbZAT), in this project. In order to functionally characterize LbZAT as an acyltransferase enzyme, proteins derived from a series of vectors harboring different LbZAT mutants will be tested. The RNAi and overexpression transgenic lines will be generated so as to characterize the LbZAT features in acyltransferase and plastid development using Omics, HPLC-MS, TEM and other methods. To investigate the plastid developmental function of LbZAT, several technologies will be adopted, including protein-protein-interaction, subcellular localization and iTRAQ. The Goji fruits will be treated with acyltransferase inhibitor in order to investigate the effect of LbZAT enzyme activity on its function in plastid development. These results will lay foundation for uncovering the molecular mechanism of LbZAT promoting the biosynthesis and storage of carotenoids, which will ultimately facilitate to provide the critical gene resource for improving new varieties of wolfberry and metabolically engineering.

宁夏枸杞(Lycium barbarum)干果俗称枸杞子，因富含“养肝明目”潜在活性物质如酯化类胡萝卜素，而成为大众青睐的养生保健品；它是迄今报道玉米黄素双棕榈酸酯(ZDP)含量最高的植物组织。尽管模式植物中类胡萝卜素合成主干路线已经清晰，但是类胡萝卜素酯化修饰促进其在果蔬中合成和存储的普遍保守分子机制尚不明晰。本项目采用交叉学科研究方法，筛选催化合成ZDP的候选基因(LbZAT)；构建LbZAT突变系列载体，鉴定其酰基转移酶功能特征；构建RNAi和超量表达转基因株系，采用组学、HPLC-MS和TEM等方法解析LbZAT的酰基转移酶和质体发育功能；采用蛋白互作、亚细胞定位和iTRAQ等技术，解析其质体发育功能；通过酰基转移酶抑制剂处理，解析LbZAT酶活对其质体发育功能的影响；为揭示LbZAT促进酯化类胡萝卜素合成存储的分子机制奠定基础。最终为枸杞新品种改良和代谢工程研究提供重要基因资源。

本项目旨在挖掘、解析枸杞子宁夏枸杞（Lycium barbarum L.）“养肝明目”功效物质合成关键基因玉米黄素双棕榈酸酯酰基转移酶基因的功能。本研究利用转录组、蛋白组等研究方法，结合WGCNA分析方法，精准挖掘、锁定到了玉米黄素双棕榈酸酯酰基转移酶基因LbZAT1和LbZAT2。组织时空表达分析表明，LbZAT1在果实中特异表达，且LbZAT1蛋白表达量与质体数量成正比、随着果实成熟而不断提高；LbZAT2在检测组织中广泛表达。亚细胞定位分析揭示，LbZAT1和LbZAT2均“点状”定位于质体。基于共表达分析结果，本研究选取了LbMinE、LbFstZ2b两个蛋白，探究其与LbZAT蛋白互作关系。酵母双杂（Y2H）表明，LbZAT与LbMinE蛋白互作；但是CoIP、BiFC初步结果表明两者没有蛋白互作关系。本研究构建了LbZAT的一系列序列缺失突变体载体，尽管尝试了密码子优化、转化大肠杆菌YLZ004菌株和酿酒酵母WAT11菌株等多种方式表达LbZAT蛋白并研究其体外功能；但是很遗憾没有表达出蛋白，究其原因可能是LbZAT编码质体膜蛋白，它的蛋白稳定性可能需要特殊的微环境。目前，本项目已经获得了LbZAT的超量表达转基因幼苗，CRISPR和RNAi转基因愈伤组织，后续将继续研究LbZAT在果实中的功能。值得注意的是，宁夏枸杞LbZAT超量表达愈伤组织材料HPLC分析表明，与对照相比，LbZAT促进了酯化类胡萝卜素的合成积累；亚细胞结构分析表明，LbZAT1/2均能促进质体数量增加。此外，在烟草叶片中瞬时共表达LbCHYB和LbZAT基因发现，与对照相比，LbZAT具有催化合成玉米黄素双棕榈酸酯的功能、且显著提高玉米黄素双棕榈酸酯含量。本项目还获得了LbZAT的超量表达转基因水稻F2种子，尽管LbZAT转基因水稻种子种皮呈现黄色，但是HPLC未检测到玉米黄素双棕榈酸酯产物，这可能与水稻胚乳中缺乏棕榈酸底物有关。本研究结果将为宁夏枸杞功效物质遗传改良提供重要的基因资源，进而服务、提升枸杞产业高质量发展。项目执行期间，共发表9篇SCI期刊论文、1篇核心期刊论文，申请专利2项；培养毕业博士生3名，硕士生3名；以本项目研究内容为基础成功申请、获批国家重点研发项目子课题1项。