丹参作为传统中草药，其活性成分丹参酮类化合物在治疗心脑血管等疾病方面均有显著活性。因此，丹参酮类化合物生物合成及调控研究一直是研究的热点。  

本研究基于实验室前期筛选丹参转录组数据获得一个在丹参根部特异性高表达的转录因子SmAP2/ERF82。在本研究中，利用实时荧光定量PCR方法验证了SmAP2/ERF82在丹参根和根韧皮部表达量极高。蛋白亚细胞定位实验表明SmAP2/ERF82定位于细胞核。转录因子自激活活性实验表明SmAP2/ERF82不具有自激活活性。

本研究通过构建SmAP2/ERF82的RNAi和过表达重组质粒，转化发根农杆菌ACCC10060，侵染丹参叶片，获得了丹参转基因毛状根。通过检测转基因毛状根中SmAP2/ERF82基因抑制和过表达效率，筛选出三个RNAi株系（82i-1/5/9）和三个过表达株系（82oe-3/5/7）。UPLC检测了转基因毛状根中丹参酮类化合物和RNAi株系中丹酚酸类化合物的含量，结果表明：与对照株系相比，在RNAi株系中，二氢丹参酮I和丹参酮I的含量降低，而在过表达株系中，二者的含量显著升高，表明SmAP2/ERF82具有正向调控丹参酮生物合成的作用；而丹酚酸类化合物在RNAi株系中无明显变化，表明SmAP2/ERF82对丹酚酸合成和代谢不具调控作用。

为进一步解析SmAP2/ERF82参与丹参酮类化合物合成的调控机制，通过实时荧光定量PCR分析转基因毛状根中丹参酮生物合成途径关键酶基因表达量的变化。与对照株系相比，SmAP2/ERF82-RNAi株系中柯巴基焦磷酸合酶1（Copalyl diphosphate synthase 1，CPS1）和细胞色素P450 CYP76AH3的表达量均显著下降，而SmAP2/ERF82-oe（过表达）株系中（除了82oe-7），异戊烯基焦磷酸异构酶（Isopentenyl pyrophosphate isomerase ，IDI1）和CPS1关键酶基因的表达量上升。通过酵母单杂交实验发现SmAP2/ERF82 与IDI1、CPS1和CYP76AH3这三个关键酶基因的启动子区结合，说明丹参SmAP2/ERF82转录因子可能通过调控丹参酮生物合成途径关键酶基因的表达调节丹参酮的生物合成。

本研究将SmAP2/ERF82-RNAi和SmAP2/ERF82-oe过表达重组质粒，转化根癌农杆菌EH105，侵染丹参叶片，获得丹参转基因组培苗，共获得一个RNAi株系（82i-6）和三个过表达株系（82oe-1/15/16）。观察发现：与对照株系（pki）相比，RNAi株系生长正常，且根系较发达；而与对照株系（pkoe）相比，过表达株系生长矮小，叶片小而皱缩，根系较稀疏。表明SmAP2/ERF82可能参与调控丹参株形发育。

为进一步验证SmAP2/ERF82在植物生长发育中的功能，将SmAP2/ERF82过表达重组质粒转化根癌农杆菌GV3101，通过花芽浸蘸法获得了过表达SmAP2/ERF82的拟南芥。观察发现，对照株系生长正常，而过表达株系生长矮小，较少抽薹，说明在拟南芥中过表达SmAP2/ERF82基因影响了拟南芥的抽薹过程，进而影响了拟南芥的生长发育。

将SmAP2/ERF82转基因毛状根分化获得的丹参苗进行RNA-Seq测序，分析转基因株系与对照株系的差异基因情况，结果发现：在RNAi株系中，赤霉素合成途径的关键酶基因GA 20-氧化酶（Gibberellin 20-oxidase, GA20ox）和GA 3-氧化酶（Gibberellin 3-oxidase, GA3ox）的表达量上调，在过表达株系中，关键酶基因GA20ox和GA 2-氧化酶（Gibberellin 2-oxidase, GA2ox）表达量下调。推测SmAP2/ERF82可能负调控赤霉素的生物合成。

以上结果表明SmAP2/ERF82通过调控丹参酮生物合成途径中关键酶基因IDI1、CPS1和CYP76AH3的表达正调控丹参酮类化合物的生物合成；同时，还可能通过负调控赤霉素的生物合成来调控植物的生长发育过程。本研究通过鉴定SmAP2/ERF82转录因子的功能，表明该基因可能作为一个关键调节因子，调控丹参中以GGPP为共同底物的丹参酮和赤霉素这两类二萜类化合物的竞争性生物合成途径，发挥其在调控丹参酮生物合成和调节丹参生长发育过程中的重要作用，对进一步解析丹参活性成分的合成与调控机制具有重要意义。

Salvia miltiorrhiza is a traditional Chinese herbal medicine, and its active ingredient tanshinone is widely used to treat cardiovascular and cerebrovascular diseases.  Therefore, the research on tanshinone biosynthesis and regulation has been a hot research area.

In this study, a transcription factor named SmAP2/ERF82 which was highly expressed at the root was selected from the transcriptome data of S. miltiorrhiza based on the previous study in our lab. The expression profile of this gene detected by real-time quantitative PCR confirmed that SmAP2/ERF82 was highly expressed in the root and the phloem of S.miltiorrhiza. Protein subcellular localization experiments showed that SmAP2/ERF82 was localized in the nucleus, and it does not have self-activating activity.

 The recombinant plasmids of SmAP2/ERF82-RNAi and over-expressed (SmAP2/ERF82-oe) were constructed and transformed into S.miltiorrhiza induced by Agrobacterium tumefaciens ACCC10060, and the transgenic hairy roots of S.miltiorrhiza were obtained. By examining the efficiency of SmAP2/ERF82 gene suppression and overexpression in transgenic hair roots, a total of three RNAi lines (82i-1/5/9) and three overexpression lines (82oe-3/5/7) were screened. We used UPLC to detect the content of tanshinone compounds in SmAP2/ERF82-RNAi and overexpression transgenic hairy roots; and salvianolic acid compounds in RNAi lines. The results showed that the content of dihydrotanshinone I and tanshinone I was decreased in the SmAP2/ERF82-RNAi lines, while the content of these two compounds was increased significantly in the SmAP2/ERF82-overexpression lines, in comparison to the control lines, indicating that SmAP2/ERF82 positively regulated the biosynthesis of tanshinones in S.miltiorrhiza. However, the content of salvianolic acids in SmAP2/ERF82-RNAi lines was not different with that in control lines, suggesting that SmAP2/ERF82 had no effect on salvianolic acid biosynthesis and metabolism.

To further analyze the regulatory mechanism of SmAP2/ERF82 in tanshinone biosynthesis, we analyzed the expression levels of key genes involved in the tanshinone biosynthetic pathway in the transgenic hairy root lines. The results showed that the expression levels of key enzyme-coding genes CPS1 and CYP76AH3 in SmAP2/ERF82-RNAi strains were significantly reduced, while the expression levels of IDI1 and CPS1 were increased in SmAP2/ERF82-overexpression lines (except 82oe-7 line). We found SmAP2/ERF82 binded to the promoter regions of IDI1, CPS1 and CYP76AH based on yeast one-hybrid analysis, indicating that SmAP2/ERF82 regulates tanshinone biosynthesis by regulating the expression of key enzyme genes in tanshinone biosynthetic pathway.

The recombinant plasmids of SmAP2/ERF82-RNAi and SmAP2/ERF82-oe were transformed into agrobacterium EH105, and then infected leaves of S.miltiorrhiza to obtain transgenic tissue culture seedlings. One RNAi lines (82i-6) and three overexpression lines (82oe-1/15/16) were obtained. Compared with the control line (pki), the growth of SmAP2/ERF82-RNAi lines was normal. However, compared with the control line (pkoe), the SmAP2/ERF82-overexpression lines grew dwarfly, with the small and shriveled leaves and the relatively sparse roots, that indicated SmAP2/ERF82 might play essential role in regulation of the morphological development of S.miltiorrhiza.

To further verify the role of SmAP2/ERF82 in regulation of the growth and development of S.miltiorrhiza, the SmAP2/ERF82 overexpressed recombinant plasmid was transformed into Arabidopsis thaliana induced by agrobacterium GV3101. The phenotype of T1 generation of the control lines was normally, while the SmAP2/ERF82 overexpressed lines grew short and were less bolting, indicating that the overexpression of SmAP2/ERF82 gene in A.thaliana affected the bolting process and the development of A. thaliana.

The seedling of salvia miltiorrhiza grown from transgenic hairy roots were used to detect the changes in gene expression between the transgenic lines and control lines based on the Illumina deep RNA sequencing (RNA-seq) strategy. The results showed that the expression levels of GA20ox and GA3ox, the key enzyme genes in the gibberellin biosynthesis pathway, were up-regulated in the RNAi lines, while the expression level of GA20ox and GA2ox were down-regulated in the overexpressed lines, suggesting that SmAP2/ERF82 may be involved in gibberellin biosynthetic pathway.

In summary, SmAP2/ERF82 plays a positive role in regulating the expression levels of key enzyme genes IDI1, CPS1 and CYP76AH3 to further regulate the biosynthesis of tanshinons in S.miltiorrhizae. In addition, SmAP2/ERF82 may regulate plant growth and development by negatively regulating the biosynthesis of gibberellin. This study identified the function of SmAP2/ERF82 transcription factor and clarified its role in the regulation of tanshinone biosynthesis and the plant development, which will facilitate to explore the produce of tanshinones by genetic engineering and the cultivation of S.miltiorrhiza in future.