蓝莓果实熊果酸合成关键酶基因的克隆与功能鉴定

Triterpenoids like ursolic acid exhibit various pharmacological activities such as antiinflammatory,.anti-tumor and anti-microbial properties. ursolic acid have also been found from Vaccinium corymbosum L in trace amounts, but the traditional breeding method is too time-consuming and difficulty to improve the ursolic acid content. Therefore, it has a great significance that exploring the biosynthetic pathway and regulatory mechanism and improving the productive efficiency of ursolic acid from the gene level. On the basis of preliminary study of our research group, Using the EST collection from Vaccinium corymbosum L leaf, we have successfully isolated a cDNA (VvAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (VvAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of VvAS and VvAO were analyzed in yeast (Saccharomyces cerevisiae) Systems. In this study, we attempt to explore new ways to improve the content of ursolic acid by verifying and regulating of key enzymes involved in its iosynthetic pathway using in vitro and in vivo methods.Then, we verify the function of the VvAS/VvAO gene by over-expression using transgenic system in vivo. Simultaneously, we clone the promoters and enhancers of VvAS/VvAO and analysis the accurate sub-cellular localization. This study will not only demonstrate the biosynthetic mechanism of VvAS/VvAO for ursolic acid, but also provide a theoretical basis and target genes for Vaccinium corymbosum L in transgenic breeding and genetic engineering.

蓝莓果实含有抗癌成分—熊果酸，但熊果酸在蓝莓果实中含量低，传统育种周期长、提高含量潜力有限。本项目以‘喜来’蓝莓果实为研究对象，通过RT-PCR和RACE技术克隆得到熊果酸生物合成途径中2个重要的调控酶—β-香树脂合成酶（VVAS）和β-香树脂氧化酶（VVAO）基因全长，在此基础上，通过体外酵母真核表达验证基因的催化功能，确定特异催化熊果酸合成的关键酶基因。通过将2个关键酶基因导入蓝莓植株进行超表达，同时进行启动子克隆及亚细胞精细定位，最终从基因表达、启动子、亚细胞定位、酶动力学及转基因等多方面详细阐明VvAS和VvAO在熊果酸合成中的分子机制，为最终实现蓝莓的遗传操作、培育高熊果酸含量的蓝莓新品种奠定基础。

蓝莓果实含有抗癌成分—熊果酸，但熊果酸在蓝莓果实中含量低，传统育种周期长、提高含量潜力有限。本项目以‘喜来’蓝莓果实为研究对象，通过RT-PCR技术克隆得到熊果酸生物合成途径中2个重要的调控酶—β-香树脂合成酶（VVAMS1）和β-香树脂氧化酶（VVAO1）基因全长，扩增获得的VvAMS1基因全长2578bp，包含开放阅读框2301bp，共编码767个氨基酸；扩增拼接后的VvAO1基因全长1792bp，包含开放阅读框1446bp，共编码481个氨基酸。利用qRT-PCR分析了蓝莓在不同发育时期（青果期和成熟期）与不同器官（根系、叶片、花、休眠芽等）的表达丰度，结果表明筛选的VvAMS1和VvAO1在花、青果和根表达量较高。并研究了茉莉酸甲酯（MJ）诱导2h、4h、8h、24h、48h基因的表达规律，MJ处理明显增加VvAMS1和VvAO1基因的表达量。构建VvAMS1和VvAO1基因的真核表达载体pYES2-VvAMS1-VvAO1和空载体pYES2-Trp分别转入酿酒酵母WAT11菌株，分析获得催化产物为熊果酸。采用Tail-PCR，克隆得到了两个基因约1500bp 长度的启动子，预测均含ABRE、LTR等基序，连接GUS在烟草中表达后验证了启动子转录活性。通过叶盘法转化蓝莓，采用HPLC技术研究发现组成型启动子35S驱动下的转VvAMS1和VvAO1基因株系的熊果酸含量明显增加。初步探讨了VvAMS1和VvAO1参与熊果酸合成的分子机制。