阳春砂龙脑基二磷酸合酶功能优化及其在种子中特异表达的解析

Bornyl acetate is the main active medicinal compound in Fructus Amomi, one Traditional Chiense Medicine product. Its content is closely relating to medicinal quality of Fructus Amomi. Previous studies have shown that this compound is rich in seeds of plants. To date, its biosynthetic pathway remains unclear. Applicants’ previous studies have identified bornyl diphosphate synthase (AvBPPS) gene from Amomum villosum. Its main product bornyl diphosphate (BPP) is the key precursor of bornyl acetate, and it is closely related with the enrichment of bornyl acetate in the seeds of Amomum villosum, however, the key amino acid sites responsible for BPPS producing special monoterpene precursor BPP have not yet been discovered. Moreover, whether AvBPPS promoter has seed specificity, which determines the specific expression of AvBPPS in seeds, also needs to be explored. This project is designed to compare AvBPPS with other BPPSs on catalytic functions and molecular modeling, then identify essential amino acids controlling the formation of BPP through amino acid site-specific mutation, and screen functionally optimized AvBPPS. The other study of this project is clone the promoter of AvBPPS and use the model plant Camelina to verify the seed specificity of AvBPPS promoter and its regulatory elements, and notarize the function of the optimized AvBPPS. The accomplishment of this project will generate understanding about the function of special monoterpene synthase BPPS, and knowledge on the mechanism of medicinal substance synthesis and enrichment in specific areas of plants, and provide molecular operation elements to improve the quality of Fructus Amomi through metabolism regulation.

乙酸龙脑酯是芳香化湿药砂仁的主要药效物质，它在药用部位种子中的富集影响砂仁的品质，而其生物合成和富集的机制尚待阐明。申请人从阳春砂中发现了龙脑基二磷酸合酶AvBPPS，其主产物龙脑基二磷酸BPP是乙酸龙脑酯的关键前体，与乙酸龙脑酯在阳春砂种子中的富集密切相关，然而，决定BPPS催化生成BPP的关键氨基酸位点未被解析，AvBPPS启动子是否具有种子特异性，从而决定其在种子中特异表达，也有待探究。本项目将AvBPPS与其他BPPS进行催化功能及分子建模比较，通过氨基酸定点突变，对生成BPP的关键位点进行实验解析，并筛选AvBPPS优化酶；克隆AvBPPS启动子，在模式植物亚麻荠中验证启动子的种子特异性及其调控元件，并确认AvBPPS优化酶的功能。本项目的实施将深入解析特殊单萜合酶BPPS的功能，丰富对药效物质在植物特定部位合成及富集机制的认识，为通过代谢调控提高砂仁药材品质提供操作元件。

乙酸龙脑酯是芳香化湿药砂仁的主要药效物质，它在药用部位种子中的富集影响砂仁的品质，而其生物合成和富集的机制尚待阐明。申请人从阳春砂（Amomum villosum）中发现了龙脑基二磷酸合酶AvBPPS，其主产物龙脑基二磷酸BPP是乙酸龙脑酯的关键前体，与乙酸龙脑酯在阳春砂种子中的富集密切相关。本项目通过分子建模、构建突变体和实验验证，初步解析了AvBPPS催化生成BPP（龙脑基二磷酸）的关键氨基酸，并获得了功能优化的突变体酶；克隆了AvBPPS的启动子，并通过启动子截短和GCN4-motif突变体转基因，验证了GCN4-motif的种子表达特异性及正调控的功能。.通过对不同植物来源的BPPS，包括阳春砂近缘植物海南砂的AlBPPS，进行体外功能比较以及分子建模和底物对接，预测了AvBPPS的关键氨基酸位点，并通过突变体进行了验证。发现了13个影响AvBPPS活性的关键位点，包括7个可能影响其产物BPP选择性的关键位点（I342、V450、Y574、L569、A507、Y424、G495），3个可能影响其产物BPP独特性的关键位点（V345、T451、F488），和3个可能影响其整体催化活性的关键位点（Q435、Y513、I351）。获得了5个BPP选择性提高的突变体，其中AvBPPS-Y513A提高到野生型的1.5倍，实现了对AvBPPS的功能优化。.获得阳春砂AvBPPS和海南砂AlBPPS的启动子，两者3’端约300bp的序列高度相似，AvBPPS启动子包含胚乳表达相关的GCN4-motif。对AvBPPS和AlBPPS的启动子进行了截短，并在此基础上对GCN4-motif进行突变，获得了相应启动子介导GUS基因表达的转基因烟草。研究结果表明，截短且含有GCN4-motif的AvBPPS启动子介导的GUS转基因植株种子中的GUS表达量高于其他器官中的表达量，GCN4-motif突变的启动子失去种子表达特异性，证明截短的AvBPPS启动子具有更强的活性，并且GCN4-motif是调控AvBPPS在种子中特异高表达的关键调控元件。.本项目从AvBPPS的催化功能和种子特异表达的角度，解析了阳春砂龙脑相关的萜类药效物质，特别是乙酸龙脑酯在药用部位种子中富集的分子机制，为通过代谢调控提高砂仁药材品质提供了操作元件。