大豆查尔酮合成酶(CHS)基因的克隆、表达及其在雪莲提取液中的代谢产物分析

以中国大豆为材料，利用PCR方法克隆查尔酮合成酶（Chalcone synthase，CHS）全基因，采用SOE法克隆得到去掉内含子的查尔酮合成酶基因，核酸序列分析表明，该基因编码区长1170bp，编码390个氨基酸，与已报道的CHS的cDNA序列同源率达到97%。构建pET-GMCHS工程表达质粒，通过大肠杆菌E.coli BL21（DE3）高效表达系统表达大豆CHS。 通过12% SDS-聚丙烯酰胺凝胶电泳分析表明，获得了分子量在42.9KD的一条蛋白质特异表达带。液相色谱分析大肠杆菌E.coli BL21（DE3）高效表达系统在雪莲提取液中的代谢产物，样品和空白对照样对比，样品在273nm，3.0min出现新的吸收峰，质谱分析结果表明CHS利用雪莲提取液中代谢中间产物合成了新的黄酮类物质。

Cloning and Expression of Chalcone Synthase(CHS) of Glycine max L and analysis of it metabolize produce in the extracts from saussurea spp

The chalcone synthase gene of Glycine max L was cloned by SOE-PCR method from the genomic DNA. The GMCHSEX was cloned into the pET- 31b in frame and expressed in E. coli. The protein was segregated by 12% SDS-PAGE gel electrophoresis after inducing by IPTG. A protein band with predicted size was detected. HPLC analysis of CHS metabolize produce in the extracts from saussurea spp, HPLC on Dikma Diamomsil Column-C18 (5μm，250×4.6mm) was employed using Vmethanol:Vwater=50:50 as the mobile phase at 1.0ml/min of flow rate. The detection wavelength was 273nm and 360nm. A new absorption peak appear at 3.0min , 273nm and there is abvious enhance of absorption peak at 9.3min, 360nm. Mass spectra proved three new flavonoid composition be synthesized.