药用植物苍耳腺体细胞特异性表达倍半萜合酶基因功能分析

As a sesquiterpenoid with great anti-tumor activities, xanthatin is mostly biosynthesized in the glandular trichomes on the surface of Xanthium sibiricum organs (it was discovered by us). Until now, the gene encoding the sesquiterpene synthase in xanthatin biosynthetic pathway has not been isolated. In this study, we will try to isolate several genes encoding sesquiterpene synthases from the isolated glandular trichomes of Xanthium sibiricum, and investigate their gene expression patterns in different tissues that include roots, shoots, leaves, and the isolated glandular trichomes. For the glandular trichome specifically-expressed genes, their full-length cDNAs will be cloned using RACE-PCR (Rapid Amplification of cDNA Ends) techniques. The functions of the genes will be analyzed biochemically in vitro through the expressions and purifications of these sesquiterpene synthases in E. coli cells or in vivo through transferring the genes into tobacco plants. The success of the project will provide the key gene resources for the regulations of xanthatin biosynthesis in Xanthium sibiricum or the biosynthesis of xanthatin in heterologous organisms, which will be important for high productions of xanthatin.

苍耳素是一种具显著抗肿瘤功效的倍半萜类物质，主要合成于苍耳植物组织表面的腺体细胞(本项目的前期研究结果)，其生物合成途径中的关键倍半萜合酶基因至今未被分离。本项目拟从苍耳组织表面腺体细胞中分离出几种倍半萜合酶基因，利用qRT-PCR技术分析它们在苍耳不同组织（根、茎、叶与分离的腺体细胞）中的空间表达模式；对于在苍耳腺体细胞特异性表达的倍半萜合酶基因，利用RACE-PCR (Rapid Amplification of cDNA Ends) 技术克隆出它们的全长cDNAs，在大肠杆菌中表达并纯化倍半萜合酶蛋白；通过体外酶促生化反应以及倍半萜合酶基因在烟草植物中的过量表达，分别在体外与体内对苍耳腺体细胞特异性表达倍半萜合酶基因的功能进行鉴定。本项目的研究成果将为调控苍耳素的生物合成或异源生物合成苍耳素提供必需的基因资源，进而将促进苍耳素的产业化规模发展。

苍耳素是一种具显著抗肿瘤功效的倍半萜化合物，主要合成于苍耳植物组织表面的腺体细胞（本项目的前期研究结果），其生物合成途径关键倍半萜合酶基因至今未被分离。本项目取得以下研究结果：以苍耳嫩叶为实验材料，成功地分离到纯度较高的苍耳腺体细胞；利用RT-PCR技术，从苍耳腺体细胞中分离了倍半萜合酶基因，并借助RACE-PCR技术克隆了此倍半萜合酶基因的全长cDNA序列；利用酵母与大肠杆菌系统，对此倍半萜合酶的生物化学功能进行了分析。