基于β-葡萄糖醛酸苷酶基因解析高含量黄芩素形成的分子机制

The special synthesis of flavonoids from Scutellaria bacalensis Georgi is that further extends from baicalin to baicalein, forming a special chemical quality marker component of Huangqin, which is the combination of baicalein and baicalin. Although baicalin is the active ingredient stipulated in the Chinese Pharmacopoeia, studies have shown that the bioavailability of baicalein is higher than that of baicalin, and it is more suitable as an important indicator for evaluating the quality of Huangqin. Applicants have found significant differences in the content of baicalein in different populations and under the same cultivation conditions, indicating that this difference is mainly determined by genetics, but the molecular mechanism of the difference in the content of baicalein has not yet been revealed. β-glucuronidase (GUS) is a key enzyme found in Huangqin that catalyzes the formation of baicalein, but has not been found in other flavonoid-containing medicinal materials. It is hypothesized that GUS is the key factor determining the difference of baicalein content in Huangqin. This project will study the relationship between GUS and baicalein content by comprehensively using chemical analysis methods, gene analysis techniques and gene expression analysis techniques, and further analyzes the molecules mechanism of high content of baicalein using the gene sequence characteristics, expression amount and enzyme catalytic efficiency. These lay a theoretical foundation for the molecular breeding of high quality Huangqin.

黄芩黄酮类化合物生物合成特殊之处是从黄芩苷进一步延伸至黄芩素，形成黄芩特殊的化学质量标志组分，即黄芩素和黄芩苷并存。虽然黄芩苷是我国《药典》规定的活性成分，但研究表明黄芩素的生物利用度比黄芩苷高，更适合作为评价黄芩药材质量的重要指标。申请者已经发现全国不同产地以及相同栽培条件下黄芩素含量都呈现显著性差异，说明这种含量差异主要是由遗传决定的，但目前黄芩素含量的分子机制尚未揭示。β-葡萄糖醛酸苷酶（GUS）是在黄芩中发现的将黄芩苷催化形成黄芩素关键酶基因，而在其他含黄酮类化合物药材中尚未发现。由此提出假说，GUS基因是决定黄芩药材中黄芩素含量差异的关键因素。本项目综合利用化学分析方法、基因分析技术和基因表达分析技术，研究GUS基因与黄芩素含量的关系，并进一步从基因序列特征、表达量和酶催化效率多重验证，解析高含量黄芩素形成的分子作用机制，为优质黄芩的分子育种奠定理论基础。

黄芩是一种重要的中国药用植物，黄芩素为黄芩中主要活性成分，生物利用度比黄芩苷高，更适合作为评价黄芩药材质量的重要指标。β-葡萄糖醛酸苷酶（GUS）是在黄芩中发现的将黄芩苷催化形成黄芩素的关键酶基因。本项目对黄芩β-葡萄糖醛酸苷酶（SbGUSs）进行功能研究，初步解析了SbGUS的表达量与黄芩素含量之间的相关性。通过转录组数据分析初步筛选了7个SbGUS基因，并利用大肠杆菌表达系统发现SbGUS1和SbGUS2具有催化黄芩苷生成黄芩素的活性。然后阐明了SbGUS1和SbGUS2基因表达量与6个产地的黄芩素含量之间的相关性,并通过重组酵母表达系统考察了SbGUS1和SbGUS2在不同产地的黄芩中的催化效率。结果表明，SbGUS1基因的表达与黄芩素的含量无关，而SbGUS2基因的表达与黄芩素的含量呈正相关，高含量组的SbGUS酶催化效率较低含量组更高。通过分析黄芩素高、低含量组的SbGUSs的序列特征，发现SbGUS1有两个可变位点，而SbGUS2有六个可变位点。本研究解析了SbGUS基因影响黄芩素含量变化的分子机制，为优质黄芩的分子标记育种奠定理论基础。