粉葛PtCHI基因的克隆、原核表达和亚细胞定位

Cloning, prokaryotic expression and subcellular localization of PtCHI gene from Pueraria montana var. thomsonii

关键词:  粉葛, PtCHI, 克隆, 原核表达, 亚细胞定位

Key words:  Pueraria montana var. thomsonii, PtCHI, cloning, prokaryotic expression, subcellular localization

摘要:

为探究葛根品种间异黄酮类物质代谢关键酶基因PtCHI的分子机制差异,并揭示其品种间异黄酮物质含量差异的原因。该研究以野葛品种‘桂葛8号'和粉葛品种‘桂葛1号'为材料,经乙醇提取并通过高效液相色谱仪对野葛和粉葛中葛根素和总黄酮的含量进行测定; 基于已报道的野葛CHI基因,通过同源克隆方法分离粉葛中PtCHI基因,并在体外进行蛋白表达,同时在拟南芥原生质体中研究PtCHI基因的定位。结果表明:(1)野葛中的葛根素含量显著高于粉葛的,野葛的总黄酮含量也高于粉葛但未达到显著水平。(2)成功分离到粉葛PtCHI基因,长度为742 bp且包含672 bp完整的ORF框,编码223个氨基酸,与野葛的CHI基因具有99%的同源性。(3)CHI基因在粉葛中的表达量为茎>根>叶子,在野葛中则为根>茎>叶子,除叶子外野葛中CHI基因的表达量均显著高于粉葛。(4)经预测,粉葛PtCHI蛋白为稳定的亲水性蛋白且大小为27.8 kD,二、三级结构以α-螺旋为主,具有25个磷酸化位点,与野葛、大豆和乌拉尔甘草的亲缘关系较近,与F3H2、F3H、4CL4、DFR2及CHS发生互作的可能性较大。(5)在体外成功诱导并分离到27.8 kD的PtCHI单一蛋白。(6)通过拟南芥原生质体进一步揭示PtCHI主要定位在叶绿体。该研究结果进一步解析了粉葛和野葛中黄酮类物质含量的差异问题,为PtCHI的功能验证和异黄酮代谢途径机理研究奠定了基础。

Abstract:

In order to explore the differences in the molecular mechanism of the Pueraria cultivars between isoflavone metabolic enzyme gene PtCHI, and to preliminarily reveal the difference content causes of the isoflavones. The materials of the study were Pueraria montana var. lobata and P. montana var. thomsonii. Puerarin and total flavonoids of P. montana var. lobata and P. montana var. thomsonii were extracted by ethanol, and their contents were measured by high-performance liquid chromatography. Based on the reported CHI gene of Pueraria montana var. lobata, the PtCHI gene from P. montana var. thomsonii was isolated by homologous cloning method, and the protein was expressed in vitro. At the same time, the location of the PtCHI gene was studied in Arabidopsis thaliana protoplasts. The results were as follows:(1)The content of puerarin in P. montana var. lobata was significantly higher than the P. montana var. thomsonii, and the content of total flavonoids was also higher but not significant.(2)The gene PtCHI was successfully isolated from P. montana var. thomsonii. The gene was 742 bp in length, containing a complete ORF frame of 672 bp, encoding 223 amino acids, and had up to 99% homology with P. montana var. lobata.(3)This study found that the expression of CHI gene in P. montana var. thomsonii was stem>root>leaf, P. montana var. lobata was root>stem>leaf. The expression of CHI gene from P. montana var. lobata was significantly higher than P. montana var. thomsonii besides in leaves.(4)Through the online tool prediction analysis, PtCHI was found to be stable hydrophilic protein and the size was 27.8 kD. The secondary and tertiary structures were based on α-helix, with 25 phosphorylation sites, closely relating P. montana var. lobata, Glycine max and Glycyrrhiza uralensis, and were more likely to interact with F3H2, F3H, 4CL4, DFR2 and CHS.(5)At the same time, the protein of PtCHI was successfully induced and isolated in vitro, with a single protein of 27.8 kD.(6)Through the Arabidopsis thaliana protoplasts revealed that PtCHI was mainly located in the chloroplasts. This study is in further analyzing the difference in flavonoids in P. montana var. lobata and P. montana var. thomsonii, as well as laying the foundation for the functional verification of P. montana var. thomsonii PtCHI and the research on the mechanism of isoflavone metabolism.