番茄茄红素β-环化酶基因高效沉默载体构建

Construction of Efficient Gene Silencing Vector Harboring Lycopene β-Cyclase Gene

番茄茄红素是番茄果实中含量最多的一种类胡萝卜素,也是植物类胡萝卜素代谢过程中的一个重要中间产物。它的环化是在茄红素β-环化酶(Lyc-b)和茄红素ε-环化酶(Lyc-e)催化下完成的,其中茄红素β-环化酶在番茄茄红素转变为β-胡萝卜素的过程中起主要作用。我们利用RT-PCR方法从番茄叶片中克隆出Lyc-b基因,将PCR产物与pGEM-T载体连接后进行测序,扩增的DNA序列与发表的茄红素β-环化酶序列一致。以此为模板,克隆了400bp左右的目的片段,利用Gateway系统构建Lyc-b基因目的片段的pJawoh18-2b ihpRNA表达载体。经PCR与酶切鉴定的阳性克隆转化农杆菌LBA1100,利用农杆菌叶盘法转化番茄获得了抗性愈伤及再生苗,以检测ihpRNA沉默载体能否降低茄红素β-环化酶基因表达,并为进一步了解该基因在番茄类胡萝卜素代谢过程中的作用奠定基础。这些数据未在本文列出。

Lycopene is the major kind of carotene in tomato fruits as well as an important intermediate in the metabolic process of carotene. Cyclization of lycopene is catalyzed by lycoene β-cyclase （Lyc-b） and lycoene ε-Cyclase （Lyc-e） and also the lycoene β-cyclase （Lyc-b） is the key enzyme in carotenoid biosythefic pathway, which catalyzes the formation of β-carotene from lycoene. In this paper we reported that the tomato Lyc-b gene was amplified by RT-PCR from tomato leaf mRNA and inserted into the pGEM-T vector. We obtained the Lyc-b gene that is the same as the published sequences. The 400 bp fragment was subcloned into vector to form pJawohl8-2b ihpRNA gene silencing vector by Gateway system approach. The pJawohl8-2b containing Lyc-b gene fragment was constructed and transformed into Agtobacterium LBA1100 validated by PCR and restriction analysis. The tomato callus and seedling were transformated via Agtobacterium-mediation in order to understand the constructs ofLyc-b gene whether is working well, these data is unpublished in this paper.