番茄红素环化酶基因片段克隆及反义表达载体构建

Cloning and Construction of Antisense Expression Vectors of Lycopene Cyclase Genes

根据已知的番茄红素环化酶基因,即β环化酶基因和ε环化酶基因的保守序列设计特异性引物。提取番茄叶片的RNA,经RT-PCR反应,扩增出723 bp和569 bp的目的片段,将它们分别连接到pEASY-T1 Cloning Vector上,测序鉴定其正确性。克隆到载体上的β环化酶基因用BamHⅠ和SmaⅠ双酶切,将基因反向插入PROK2表达载体上,构建PROK2-LYCb反义表达载体。同样连接到克隆载体上的ε环化酶基因用BamHⅠ和XbaⅠ双酶切,将基因反向插入PROK2表达载体上,构建PROK2-LYCe反义表达载体。

Based upon the known lycopene cyclase genes β and e, specific primers were designed. The RNA extracted from tomato leaves was used for RT- PCR, then the 723 bp and 569 bp gene fragments were amplified and were subcloned into pEASY -T1 Cloning Vectors for sequencing. LYCb gene was digested by BamH I and Sma I and inserted into PROK2 expressing vector to construct the antisense vector PROK2 - LY- Cb. In the same way,LYCe gene was digested by BamH I and Xba I and inserted into PROK2 expressing vector to construct the antisense vector PROK2 -LYCe.