决明查尔酮合成酶全长cDNA的克隆及其原核表达载体的构建

Cloning and Prokaryotic Expression Vector Construction of Cassia tora Chalcone Synthase

从决明子叶中提取查尔酮合成酶(Chalcone synthase)总RNA,经过RT-PCR获得查尔酮合成酶cDNA,纯化后与pMD18-T载体连接,转化大肠杆菌Top10,获得了查尔酮合成酶全长基因序列。序列测定表明,查尔酮合成酶全长核苷酸长度为1173 bp,编码390个氨基酸。与GenBank中已发表序列EU430077进行比较,核苷酸同源性为100%。将该基因片段克隆到原核表达载体pET-28a(+)中,构建重组质粒pET-28a(+)/Chalcone synthase,所获重组质粒经过双酶切、PCR和序列测定,证实含有目的片段,且连接、构建正确,为查尔酮合成酶的进一步表达奠定了基础。

The total RNA of chalcone synthase was extracted from leaf of Cassia tora and its cDNA was obtained by RT-PCR. The purified RT-PCR products and pMD18-T vector were ligated and transformed into host strain E. coli Topl0. The full length of Chalcone synthase gene is consisted of I 173 bp,which encodes 390 amino acids. The homology of the nucleotides with EU430077 is 100%. The aim gene was expression on the prokaryotic expression vector pET-28a（ ＋ ）and a recombinant plasmid pET-28a（ ＋ ）/Chalcone syn- thase was constructed successfully.