黄芩黄酮合成相关R2R3-MYB转录因子可变剪切异构体的功能及机理研究

Scutellariae radix is an important traditional Chinese medicine. Its flavonoids have anti-inflammatory, antibacterial and other pharmacological activities. Studies have shown that Alternative splicing (AS) plays a vital role in transcriptional gene function；R2R3-MYB transcription factors are involved in the synthesis of Scutellariae flavonoids. Hybrid sequencing of S. baicalensis has been completed by our team. Based on annotation and coexpression analysis with key enzyme genes, one flavonoid synthesis related R2R3-MYB transcription factor with AS events were screened, then its three isoforms were validated by PCR. what are the differences between three R2R3-MYB transcription factor isoforms in S. baicalensis, which one has stronger activity and function, which one plays a dominated role in regulating the synthesis of Scutellariae flavonoids, these issues still need to be studied. This study was designed to analyze the structure and phylogenetic relationships of these three AS isoforms, investigate their expressions and perform their subcellular localization. In addition, construct the over-expression and RNAi vector with the AS genes respectively. Transform Arabidopsis thaliana and the hairy roots of S. baicalensis. By detecting the expression of the target genes, the contents of flavonoids in transgenic plants, and expression profiles of key enzymes related to flavone synthesis and three isoforms genes in S. baicalensis, validate the functions of the AS genes and determine the dominant AS isoform. In a word, the function and mechanism of the AS genes of R2R3-MYB transcription factor involved in regulation of flavonoids biosynthesis of S. baicalensis will be preliminarily clarified, which will lay the foundation for the variety breeding, genetic improvement, increasing flavonoid production, and synthetic biology of S. baicalensis.

黄芩是我国重要传统中药，其黄酮类物质具有抗炎、抗菌等药理活性。研究显示可变剪切在调控基因功能方面具有重要作用；R2R3-MYB转录因子黄芩参与黄酮类化合物的合成。前期已完成黄芩二、三代联合转录组测序，根据注释及共表达分析筛选了具有可变剪切的黄芩黄酮合成相关R2R3-MYB转录因子基因，且对3个异构体进行了验证。它们有何差异，哪种具更强活性与功能，在调控黄芩黄酮合成中发挥关键作用，这些问题亟待研究。本研究拟分析3个异构体的结构及进化关系，考察其表达情况并进行亚细胞定位。构建不同异构体基因的过表达和RNAi载体，侵染拟南芥同时转化黄芩毛状根，通过检测转基因植物的黄酮含量及分析黄芩黄酮合成相关关键酶、异构体基因的表达情况，鉴定不同异构体的功能，确定主导剪切类型，初步解析R2R3-MYB转录因子可变剪切在调控黄芩黄酮合成的作用。研究将为黄芩品种改良、提高黄酮产量及合成生物学提供可能。

传统中药黄芩的主要活性成分黄酮类物质具有抗病毒、抗炎等药理活性，但是其天然产物含量有限。本项目筛选得到的参与黄芩黄酮类化合物合成相关的转录因子SbMYB13，并对其进行了可变剪切及功能研究。研究显示：SbMYB13存在三种不同的可变剪切类型：A型属于正常编码类型；B型属于短的内含子保留类型（Short IR type）；C型属于长的内含子保留类型。通过生信预测及荧光定量PCR验证发现：A、B、C型均在叶中表达量最高，其次是根，最后是茎。无论在哪种部位，A型均为主要类型，占据绝对优势，约为BC型的2倍及以上；B-型和C型相差不大。接下来对其生物信息学以及功能分析A型剪切类型进行。亚细胞定位显示SbMYB13蛋白定位于细胞核。过表达SbMYB13基因（A型）的拟南芥纯合株系中AtCHS、AtCHI基因表达量以及总黄酮含量均显著低于野生型，VIGS病毒介导的SbMYB13基因（A型）沉默株系的黄芩叶片中SbCHS、SbCHI、SbF6H基因的表达量及其总黄酮含量均显著高于对照组。实验结果表明，SbMYB13基因（A型）负向调控黄酮类化合物的生物合成。本研究为黄芩品质改良以及分子育种提供了重要的理论基础。