(-)-BlBPPS基因及其突变体在艾纳香L-龙脑生物合成中的协同调控机制

Blumea balsamifera is the main raw materials for extracting natural borneol (Ai pian). The main component of the volatile oil from B. balsamifera is L-borneol, a typical bicyclic monoterpene. Two (-)-Bornyl pyrophosphate synthase ((-)-BlBPPS) candidate genes and their deletion mutants that were specifically expressed in leaves have been obtained from B. balsamifera in our pre-study. In this project, three-dimensional structure of candidate genes and their mutants will be predicted through homology modeling techniques and the function will be verified by prokaryotic expression and RNAi techniques. qRT-PCR and Western-blot methods will be used for detecting the mRNA and protein level of (-)-BlBPPS and the mutants in response to exogenous stress and spatiotemporal variation and the coordinated regulation mechanism involved in L-borneol synthesis. To analyze the formation mechanism of the difference in L-borneol content and explore the possibility of early selection of high quality and genetically stable materials from B. balsamifera germplasm , (-)-BlBPPS novel mutation type will be excavated from different L-borneol content B. balsamifera germplasm, and the mutants number, mRNA expression level and enzyme protein content will be compared in our research. This project will provide theoretical foundation for the quality breeding and high-yield cultivation techniques of B. balsamifera.

艾纳香Blumea balsamifera(L.)DC.是提取天然冰片（艾片）的主要植物原料，其挥发油的主要成分L-龙脑，是典型的双环单萜类物质。项目前期已获得2个叶片特异表达的(-)-龙脑焦磷酸合酶（(-)-BlBPPS）候选基因及其缺失突变体。本研究拟通过同源建模技术预测候选基因及其突变体三维结构，原核表达和RNAi技术验证其功能；采用实时荧光定量PCR（qRT-PCR）和Western-blot的方法研究(-)-BlBPPS基因及其突变体对外源胁迫和时空变异的响应规律及其对L-龙脑生物合成的协同调控机制；通过挖掘不同L-龙脑含量的艾纳香资源中(-)-BlBPPS基因新的突变类型，比较其突变体数量、mRNA表达量和酶蛋白含量，解析L-龙脑含量差异的形成机制，探索对高品质且遗传稳定的艾纳香资源早期筛选的可能性。本研究将为艾纳香的品质育种和高产栽培技术提供理论依据。

摘要：艾纳香Blumea balsamifera(L.)DC.是提取天然冰片（艾片）的主要植物原料，其挥发油的主要成分L-龙脑，是典型的双环单萜类物质。本项目在前期挖掘到2个叶片特异表达的(-)-龙脑焦磷酸合酶（(-)-BlBPPS）候选基因及其缺失突变体的基础上，重点对其结构与功能进行了分析和验证，结果表明突变基因的缺失位置主要在该基因的功能区域，其中mono-TPS1基因具有L-龙脑生物合成的功能，并将其命名为(-)-BlBPPS，而突变基因不具备该功能。另外，为研究不同L-龙脑含量的艾纳香种质资源形成的机制，本研究还对艾纳香的资源分布以及最适区域的发展趋势进行分析和预测，同时对资源进行了收集、评价与筛选，建立了高L-龙脑含量艾纳香资源的识别指标。通过对(-)-BlBPPS基因及其突变体的时空表达定量分析表明，艾纳香中L-龙脑含量与(-)-BlBPPS-W的表达量直接相关，与其突变体的表达量相关性不明显。(-)-BlBPPS-W与其突变体之前的表达量如何平衡还有待进一步研究。