基于月月粉转录组测序的RcEGS和RcAAT基因在花香代谢途径上的功能研究

Rose scent is not only an important ornamental trait, but also serviced economically value as a source of natural fragrance and flavour. Due to the limit of traditional cross-breeding and the complexity of scent-related metabolic pathway, fragrance seems to have been largely lost during the later stages of breeding process.In our previous study,using the high-throughput transcriptome sequencing technology, 89,614 Unigenes from Rose chinensis 'Pallida' were obtained, among which 307 Unigenes were invovled in scent biosynthesis. Some scent-related genes were selected to analyze expression by qRT-PCR, and RcEGS and RcAAT were found many expressed in stamens and petals of R. chinensis 'Pallida',respectively. According to the above achievements, the current study will perform expressional analysis of RcEGS and RcAAT in different flower developmental stages by qRT-PCR, and test the volatile compounds,respectively. Furtherly, the full-length cDNA of RcEGS and RcAAT will be obtained by RACE technology.Then, prokaryotic expression vector pDEST17-RcEGS and pDEST17-RcAAT will be constructed by Gateway cloning technology to get heterologous recombinant protein expression in E.coli and purification. Next,the functions will be identified by in vitro enzyme activities of RcEGS and RcAAT in the presence of substrates coniferyl acetate and geraniol,and by GC/MS analysis,respectively. Finally,plant expression vectors of RcEGS and RcAAT will be constructed, and their functions will be furtherly performed by genetic transformation to roses and analysis of genetially modified roses. These results can not only lay a foundation for the research of synthesis pathway of key synthase genes in rose, but also provide candidate genes for rose-scent breeding.

月季花香不仅是重要的观赏性状，而且具有重要的经济价值。由于传统杂交育种的局限性、花香代谢途径复杂等原因，使得大多数现代月季不具有香味。项目组在前期研究中对月月粉进行了转录组测序，从中选取了花香关键酶基因进行不同组织部位的表达分析，获得了主要在雄蕊、花瓣中表达的基因RcEGS和RcAAT。在此基础上，本项目拟采用qRT-PCR方法，对RcEGS和RcAAT基因进行月月粉花不同发育时期表达分析；采用RACE方法克隆全长cDNA；利用Gateway克隆技术构建原核表达载体,获得重组表达蛋白并进行蛋白纯化，通过体外酶与底物特异反应进行酶活性鉴定及GC/MS分析；构建植物表达载体并转化月季，获得转基因植株并进行其香味成分分析，进一步明确RcEGS和RcAAT基因参与合成的香味成分。研究结果不仅有助于探明RcEGS和RcAAT基因在花香合成代谢途径上的作用，还可为培育具有香味的月季品种提供候选基因。

月季花香不仅是重要的观赏性状，而且具有重要的经济价值。由于传统杂交育种的局限性、花香代谢途径复杂等原因，使得大多数现代月季不具有香味。项目组在前期研究中对月月粉进行了花香相关的转录组测序，获得了主要在雄蕊、花瓣中表达的基因 RcEGS1 和 RcAAT1。在此基础上，本项目采用qRT-PCR方法，完成了RcEGS1和RcAAT1基因进行表达谱分析，发现这2个基因在花蕾期没有表达，随着花朵的开放表达逐渐增强，盛开期达到最强，而随着花朵的凋谢，又降低到基点，这和月季花香的释放规律一致；采用RACE方法，从中国古老月季‘月月粉’中克隆了丁香酚合成酶基因RcEGS1（GenBank No. JQ522949）、香叶醇乙酰基合成酶基因RcAAT1的全长cDNA，其中，RcEGS1全长为 1 171 bp，开放阅读框（ORF）951bp，编码 317 个氨基酸；RcAAT1的全长cDNA有 1641bp的核苷酸序列，包含1个68bp的5’-UTR，1个1371bp的OFR，1个202bp的3’-UTR;采用Gateway技术构建原核表达载体pDEST17-RcEGS1和pDEST17-RcAAT1，获得了原核表达蛋白，Western-blot 分析发现RcEGS1蛋白在花瓣和雄蕊表达，体外酶活分析结果表明RcEGS1以乙酸松柏酯为底物，参与合成丁香酚；构建RcEGS1和RcAAT1植物高表达载体并分别获得了56和31个转基因月季，通过SPME-GC/MS技术定性定量分析发现，丁香酚在转基因及未转化的月季花瓣中的含量差别明显，在转基因花瓣中的相对含量是8.36%，而对照中是3.88%；建立了病毒诱导的基因沉默（VIGS）结合嫁接技术用于月季花香基因功能快速分析的方法，利用该方法对RcEGS1 和RcAAT1基因进行了沉默，沉默植株分析发现丁香酚的相对含量由11.9%降到了4.3%；香叶醇的相对含量由4.4%降到了1.3%；研究结果一致表明RcEGS1 和RcAAT1分别参与月季丁香酚和香叶醇的合成。项目建立了月季花香基因功能快速分析技术体系，探明了RcEGS1 和 RcAAT1 基因在花香合成代谢途径上的作用。研究结果不仅为培育具有香味的月季新品种提供了候选基因，也为其它物种的花香基因研究提供了理论和技术借鉴。