中国科学院水利部水土保持研究所机构知识库

作为重要的传统中药材之一，丹参（Salvia miltiorrhiza Bunge）的市场需求量
逐年递增，同时作为“模式药用植物”，其次生代谢调控受到了广泛的关注。丹参
干燥根中丹参酮类与酚酸类成分的含量是评价药材品质的重要指标。因此，研究 其有效成分的代谢调控机制具有着重要的意义。赤霉素（GA）在调控植物生长与 发育过程发挥着十分重要的作用，虽然有研究表明 GA 处理可以显著提高丹参毛 状根中丹参酮类与酚酸类的含量，但其调控机制并不清楚。植物特有的 GRAS 蛋 白家族作为 GA 信号通路的关键调控因子，已被报道参与调控了 GA 信号通路、
根的生长发育和抗逆等很多生物过程，但其在次生代谢调控中的作用仍不清楚。 因此，本研究以丹参毛状根为材料，分析了5个SmGRAS基因对丹参毛状根生长， 以及丹参酮、酚酸类和 GA 物质合成过程中的调控作用，并进一步分析了其促进 丹参酮合成的调控机制。得到以下主要结果： 1. 分析了 GA 处理野生型丹参毛状根后，毛状根的干鲜重、丹参酮类、酚酸 类物质含量均显著升高。对 GA 处理的毛状根与对照组进行转录组分析，发现 GA 主要调控了次生代谢过程，Mapman 对次生代谢路径差异基因进一步深入分析表 明，次生代谢合成路径的差异基因大部分是 GA 诱导的，尤其是莽草酸途径、MVA 途径、酚类、甜菜碱类、蜡质和花青素类代谢途径。还分析了 GA 信号通路中的 差异基因，发现 GA 受体 GID1 与部分 SmGRAS 家族基因表达水平都发生了显著 的升高。 2. 全基因组鉴定了35个SmGRASs家族基因，并对其进行了生物信息学分析。 系统进化树分析显示 SmGRASs 家族蛋白聚为 10 个亚家族。氨基酸序列比对显示 35 个 SmGRASs 蛋白均具有保守的 GRAS 结构域，但不同亚家族的 SmGRASs 蛋 白具有不同的基序，因此可能发挥着不同的功能。进一步分析了 35 个 SmGRASs 基因的结构组成和理化性质。另外，转录组数据的筛选结果发现大部分 SmGRASs 的转录水平受到 GA 的显著影响。 3. 在丹参毛状根中筛选并克隆了 5 个对 GA 响应显著的 SmGRAS 基因，分析 了 SmGRAS1~5 的组织特异性表达模式，发现他们在根周皮的表达量是最高的， 在其他部位的表达略有差异。烟草原生质体的亚细胞定位显示 SmGRAS1~5 蛋白
定位在细胞核，可能作为转录因子行使调控功能。转录自激活实验表明SmGRAS1/3 具有转录自激活活性，SmGRAS2/4/5 则不具有。SmGRAS1~5 的启动 子元件分析发现它们都含有 GA 响应元件，还有其他激素（ABA、MeJA 和 Aux）、 光响应与非生物胁迫的响应元件。在 GA、MeJA、ABA、NAA 和 SA 处理下，大 部分的 SmGRAS1~5 的表达量均有显著的变化，但在时间和趋势上略有差异，表 明其在激素信号通路中具有不同的功能。 4. 分别获得 SmGRAS1~5 的过表达与反义表达毛状根，发现 SmGRAS1~5 的
过表达毛状根出现了生长受抑制的情况，其干鲜重均显著降低。分别过表达 SmGRAS1~5 可以显著提高丹参酮类的含量，降低 GA 的含量。但是对酚酸类的调 控是不同的，过表达 SmGRAS1/2/3/5 降低了酚酸的含量，而过表达 SmGRAS4 提 高了酚酸的含量。SmGRAS1~5 反义表达株系中的成分变化趋势与过表达株系中相 反。进一步分析丹参酮、GA 和酚酸合成路径基因的表达，结果显示大部分基因
表达量的变化与成分含量结果相一致，其中丹参酮合成路径下游合成酶基因 SmKSL1 的变化是最显著的，其次还有 SmCPS1。 5. 酵母单杂交结果显示 SmGRAS1/3/4/5 可以与 SmKSL1 启动子区域结合， EMSA实验进一步证明SmGRAS1/3/4/5可以与SmKSL1启动子区域的GARE-motif 元件结合。Dual-LUC 实验表明，SmGRAS1/3/4/5 可以激活 SmKSL1 启动子的表 达，说明 SmGRAS1/3/4/5 可以通过直接激活丹参酮合成路径下游关键酶基因 SmKSL1 的表达，从而促进丹参酮的合成。 本研究发现了 SmGRAS1~5 在调控丹参毛状根生长与调控丹参酮、GA 与酚
酸类合成过程中的作用，并分析了它们的生物信息学、表达模式、诱导表达、靶 基因的互作等功能。

As one of the important traditional Chinese medicines, Danshen (Salvia miltiorrhiza Bunge) has an increasing market demand year by year, and is also a "model medicinal plant". The content of tanshinones and phenolic acids in its dried roots is an important index to evaluate the quality of medicinal materials. Therefore, it is of great significance to study the metabolic regulation mechanism of its active components. GA plays an important role in the regulation of plant growth and development. Although studies have shown that GA treatment can significantly increase the content of tanshinones and phenolic acids, its regulatory mechanism is not clear. As a key regulator of the GA signaling pathway, plant-specific GRAS protein family has been reported to participate in the regulation of GA signaling pathway, root growth and development, stress tolerance and many other biological processes, but its role in the regulation of secondary metabolism is still unclear. Therefore, in this study, the hairy roots of S. miltiorrhiza was used as the material to analyze the regulatory effects of five SmGRAS genes on the growth of hairy roots and the biosynthesis process of tanshinones, phenolic acids and GA, and the regulatory mechanism of promoting the biosynthesis of tanshinones was further analyzed. The following main results are obtained: 1. After GA treatment of wild-type hairy roots of S. miltiorrhiza, the dry and fresh weight, tanshinones and phenolic acids contents of hairy roots were significantly increased. Transcriptome analysis of GA treated hairy roots and control. It found the GA mainly control the secondary metabolism process, Mapman's further analysis of genes with secondary metabolic pathway differences shows that most of the differentially expressed genes (DEGs) in secondary metabolism biosynthesis pathway are induced by GA, especially the shikimic acid pathway, MVA pathway, phenols, betaine, wax, and anthocyanins. DEGs in the GA signaling pathway were also analyzed, and it was found that the expressions of GA receptor GID1 and most of GRAS families genes were significantly increased. 2. Thirty-five SmGRASs family genes were identified in genome-wide and bioinformatics analysis was carried out. Phylogenetic tree analysis showed that the SmGRASs family was divided into 10 subfamilies. Amino acid sequence alignment showed that all 35 SmGRASs proteins had conserved GRAS domains, but SmGRASs proteins of different subfamilies had different motifs, so they might play different functions. The structural composition and physicochemical properties of 35 SmGRASs genes were further analyzed. In addition, the transcriptome data screening results showed that the transcription level of most SmGRASs was significantly affected by GA. 3. Five SmGRAS genes with a significant response to GA in the hairy roots of S. miltiorrhiza were screened and cloned, and the tissue-specific expression patterns of SmGRAS1~5 were analyzed. It was found that all of them had the highest expression in the root periderm and some different expressions in other parts. Subcellular localization of tobacco protoplasts suggests that SmGRAS1~5 proteins are located in the nucleus, and may function as transcription factors. Transcriptional self-activation experiments showed that SmGRAS1/3 had transcriptional self-activation activity, while SmGRAS2/4/5 did not. Analysis of promoter elements in SmGRAS1~5 showed that they all contained GA response elements, as well as other hormones (ABA, MeJA and Aux), light response and abiotic stress response elements. Under the treatment of GA, MeJA, ABA, NAA and SA, the expression levels of most SmGRAS1~5 varied significantly, but there were differences in time and trend, indicating that SmGRAS1~5 had different functions in the hormone signaling pathway. 4. The SmGRAS1~5 overexpression and antisense expression hairy roots were obtained respectively. It was found that the growth of the SmGRAS1~5 overexpression hairy roots was inhibited, and the dry and fresh weight were significantly reduced. Overexpression of SmGRAS1~5 can significantly increase the content of tanshinones and decrease the content of GA. However, the regulation of phenolic acids was different. Overexpression of SmGRAS1/2/3/5 reduced the phenolic acids content, while the overexpression of SmGRAS4 increased the phenolic acids content. In SmGRAS1~5 antisense expression lines, the variation trend of components was reversed to the overexpression lines. Analysis of the expression of tanshinone, GA and phenolic acid biosynthesis pathway genes showed that most of the changes in gene expression were consistent with the results of composition content. Among them, the synthase gene SmKSL1 in the downstream of the tanshinones biosynthesis pathway was the most significant change, followed by SmCPS1. 5. Yeast one hybridization results showed that SmGRAS1/3/4/5 could bind to the SmKSL1 promoter region, and the EMSA experiment further demonstrated that SmGRAS1/3/4/5 could bind to the GARE-motif elements in the SmKSL1 promoter region. Dual-LUC experiments showed that SmGRAS1/3/4/5 could activate the expression of SmKSL1 promoter, indicating that SmGRAS1/3/4/5 could promote the biosynthesis of tanshinones by directly activating the expression of SmKSL1, which is the key synthase gene in the downstream of the tanshinones biosynthesis pathway. This study found the role of SmGRAS1~5 in regulating the growth of hairy roots, the biosynthesis of tanshinones, GA and phenolic acids, and analyzed their bioinformatics, expression patterns, induced expression and target gene interactions.