丹参（Salvia miltiorrhiza Bunge.）为我国常用大宗药材，其有效成分——丹参酮和丹酚酸类化合物具有多种药理活性，以丹参为君药的“复方丹参滴丸”在治疗心脑血管疾病方面已取得显著疗效。近年来，丹参活性成分生物合成途径分子机制已获得深入解析，但其生物合成的调控机制研究尚十分匮乏。AP2/ERF转录因子在多种药用植物中具有调控活性成分（如倍半萜青蒿素、二萜紫杉醇等）生物合成的功能，而在丹参中调控活性成分合成的AP2/ERF尚未报道。本次研究的主要研究结果如下：

1. 本课题组已获得丹参基因组图谱，注释到170个AP2/ERF转录因子，命名为Sm001-Sm170。本研究基于已有丹参不同器官（根、茎、叶、花）及根不同组织（周皮、韧皮部、木质部）的转录组数据，参照已鉴定参与丹参酮及丹酚酸类化合物生物合成关键酶基因的表达谱，筛选出两个AP2/ERF转录因子，皮<韧皮部和木质部的规律；Sm082具有在根中显著性高丰度表达的特点。通过实时荧光定量PCR实验验证这两个AP2/ERF基因的表达谱，结果与转录组数据一致。构建瞬时转化体系，侵染烟草叶片，分析Sm008和Sm082的亚细胞定位情况，证明两个转录因子定位于细胞核中。

2. 本研究构建了丹参遗传转化体系，对Sm008和Sm082进行过表达、RNAi抑制表达分析，获取了丹参转基因毛状根。检测阳性转基因毛状根中Sm008基因抑制表达和过表达的效率，筛选出3个抑制表达株系：8i-1/3/6；3个过表达株系：8oe-1/8/9。检测了转基因毛状根中丹酚酸类化合物的含量变化，每个株系做三个生物学重复。结果表明，与对照组相比，3个抑制表达株系中丹酚酸B的含量有明显下降，同时3个过表达株系中丹酚酸B含量明显上升。另外，观察到Sm008转基因毛状根和培养液呈现丹参酮类化合物所具有的黄/红色，所以对丹参酮的含量变化进行检测，结果发现，与对照组相比，Sm008抑制表达的2个株系中二氢丹参酮Ⅰ、隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA的含量在毛状根及培养液中均下降，说明Sm008基因可能对丹酚酸及丹参酮类化合物的生物合成均具有调控作用。进一步分析转基因毛状根中参与丹参酮和丹酚酸生物合成基因的表达量变化，预测转录因子可能调控的目的基因，结果表明，Sm008可能调控的丹酚酸途径的目的基因为4CL2基因，丹参酮途径的目的基因为CPS1、KSL1、CYP76AH1基因。

3. 对于Sm082的转基因株系，筛选出4个抑制表达株系：82i-1/5/7/19；1个过表达株系：82oe-9。化学含量分析结果表明，与对照组相比，4个抑制表达株系中二氢丹参酮Ⅰ、隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA明显下降，过表达株系中二氢丹参酮Ⅰ、隐丹参酮含量上升；而丹酚酸B含量在过表达及RNAi株系中均无明显变化；说明Sm082基因对丹参酮类化合物的生物合成具有调控作用，对丹酚酸类化合物的生物合成不具有调控作用。分析关键酶基因的表达量变化说明Sm082可能调控丹参酮途径的目的基因为CPS1、KSL1基因。本次研究还获得了Sm082转基因毛状根对应的转基因组培苗，观察根系及叶片的荧光情况及qRT-PCR实验证明转基因组培苗依然携带GFP报告基因且保持了Sm082基因的抑制/过表达状态。

4. 构建Sm008和Sm082基因的原核表达体系，Sm008在异源表达体系中可以表达并获得可溶性蛋白，而Sm082在蛋白提取的上清和沉淀中均未诱导表达获得蛋白。同时，我们还预测了丹参酮及丹酚酸类化合物生物合成途径中一系列关键酶基因的启动子区，筛选出含有GCC box的启动子序列并进行启动子区克隆，已克隆到启动子区的基因有参与丹酚酸类合成的SmRAS1和SmTAT1及参与丹参酮合成的SmCPS1。

实验结果初步证明Sm008和Sm082转录因子对丹酚酸及丹参酮生物合成具有调控作用，调控的分子机制仍有待于进一步解析。

Salvia miltiorrhiza Bunge. is one of the important medicinal plants and used widely as the traditional Chinese medicine (TCM). Tanshinone and salvianolic acid are the major bioactive ingredients, possessing various pharmacological activities. The medicine “Danshen dripping pills” which contains S.miltiorrhiza as the major medicinal material has a remarkable curative effect on cardiovascular and cerebrovascular diseases. The biosynthetic pathways of tanshinone and phenolic acid compounds in S.miltiorrhiza have been studied extensively, but the mechanism of regulation is still largely unknown. AP2/ERF transcription factors have been verified to be involved in the regulation of the bioactive ingredients in various medicinal plants, including sesquiterpenes artemisinin, diterpene paclitaxel, etc. However, the AP2/ERF playing roles in regulation of bioactive compound biosynthesis in S.miltiorrhiza has not been identified. The main results of this research are as below:

1. The genome of S.miltiorrhiza has been completely sequenced in the previous study, and 170 AP2/ERF transcription factors have been identifed, named Sm001-Sm170. Based on the transcriptome data of different organs (root, stem, leaf and flower) and root tissues (pericarp, phloem and xylem) of S.miltiorrhiza, two AP2/ERF genes were selected. The expression profile of Sm008 was consistent with that of SmRAS1 gene, which participates in the biosynthesis of phenolic acid compounds; while Sm082 was highly expressed in the root. The expression profiles of two AP2/ERF genes were also verified by qRT-PCR method. The nucleus-subcellular localization of these two putative AP2/ERF transcription factors were detected by the transient transformation of tobacco.

2. In this study, the genetic transformation system of S.miltiorrhiza has been constructed, and the transgenic hairy roots of S.miltiorrhiza have also been obtained. To analyze the efficiency of these two genes transformed into hairy roots of S.miltiorrhiza, the expression level of Sm008 was detected in the transgenic hairy roots of the over-expression lines and RNAi lines. Totally, three RNAi lines (8i-1/3/6) and over-expression lines (8oe-1/8/9) of Sm008 were selected. Compared with the control line, the content of salvianolic acid B was decreased in the three RNAi lines and was increased in the three over-expression lines of Sm008. In addition, the color of Sm008 transgenic root hairy and the liquid culture medium looks like the color of tanshinone compounds. Therefore, the content variation of tanshinone in the hairy roots and liquid culture medium were tested for the RNAi transgenetic lines of Sm008. The results showed that the contents of dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA were also decreased in both hairy roots and culture medium of two RNA lines (8i-1/3), compared with the control line. The results indicated that Sm008 gene could regulate the biosynthesis of tanshinone and salvianolic acid compounds. The expression level of genes in the biosynthesis of tanshinone and salvianolic acid in transgenic hairy roots were analyzed in order to predict the target genes of transcription factor. The results showed that Sm008 may regulate 4CL2 in the biosynthesis of the salvianolic acid, or regulate CPS1, KSL1 and CYP76AH1 in the biosynthesis of tanshinone.

3. Four RNAi lines (82i-1/5/7/19) and one ove-expression line (82oe-9) of Sm082 were selected. The contents of dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA were decreased in the four RNAi lines of Sm082 and the contents of dihydrotanshinone I and cryptotanshinone were increased in the overexpression line of Sm082. Meanwhile, the content of salvianolic acid B have not been effected by the expression level of Sm082 in transgenic hairy roots. The results indicated that Sm082 gene regulate the biosynthesis of tanshinones. The analysis of expression level of genes in the biosynthesis of tanshinone showed that Sm082 may regulate CPS1 and KSL1. In this study, the transgenic plantlets corresponding to the Sm082 transgenic hairy roots were also obtained. The fluorescence of the roots and leaves of the transgenic plantlets and the gene expression were detected. The results showed that the transgenic plantlets still carry the GFP reporter gene and maintained the inhibition / over-expression of Sm082 gene.

4. The heterologous expression of Sm008 and Sm082 in the E.coli system was carried out. Only Sm008 can be detected as soluble protein. Sm082 was not induced in the supernatant and precipitation. Furthermore, the GCC box was identified from the promoter of the key genes encoding enzymes involved in the biosynthetic pathway of tanshinone and phenolic acid compounds.The promoter of SmRAS1 and SmTAT1, both of that are involved in phenolic acid compound biosynthesis, and the promoter of SmCPS1, that participates in tanshinone biosynthesis, were cloned to be used for the target gene identification for these two transcription factors.

The results showed that Sm008 and Sm082 transcription factor played an important roles in regulation of salvianolic acid and tanshinone biosynthesis. The molecular mechanism of the function for these two genes need to be explored and confirmed in the following work. This study provides the basic data for illustration on the regulation mechanism for bioactive compound biosynthesis and accumulation in S.miltiorrhiza.