柿果ACC合成酶基因的克隆与植物表达载体的构建

Persimmon ACC synthase gene cloning and its plant expression vector construction

以柿果组织中分离的总RNA为模板,经RT-PCR扩增到约1.1 kb的富有柿果ACC合成酶(Fuyu-ACS)的 基因片段,将此片段克隆到pGEMR-T easy vector上,经测序分析与Genbank中平核无柿果的DK-ACS1基因核苷酸 序列的同源性为99％。设计了2对带限制性内切酶位点的特异性引物,以测序质粒为模板,PCR扩增到2个ACS- Fuyu片段。2个片段经双酶切消化后,分别以正反2个方向插入到植物表达载体pBI121的35 S启动子和NOS终止 子之间,构建成Fuyu-ACS基因的植物表达载体。

An amino-cyclopropane-1-carboxylie acid synthase(ACS) gene was amplified by Reverse Transcription Poly-merse Chain Reaction (RT-PCR) from ripening persimmon fruit. A 1 178 bp PCR product (Fuyu-ACS) was cloned. Sequence analysis showed that Fuyu-ACS nucleotide sequence was 99% identity with DK-ACS1 in GenBank. Two pairs of primers containing restriction enzyme site were designed and were used to amplify sequenced plasmid. Two PCR products were digested by the corresponding restricted enzymes respectively, and inserted between the CaMv 35 S promoter and NOS terminator of expression vector pBI121.