《植物生理学报》 2013, 49(12): 1339-1346

丹参转录因子基因SmMYC amiRNA表达载体的构建及其对丹参的转化

王浩如¹, 王健^1,2, 王仕英¹, 王喆之^1,*

¹教育部药用植物资源与天然药物化学重点实验室, 西北濒危药材资源开发国家工程实验室, 陕西师范大学生命科学学院, 西安710119; ²安康学院农学与生命科学学院, 陕西安康, 725000

通信作者：王喆之；E-mail: zzwang@snnu.edu.cn

摘　要：

丹参(Salvia miltiorrhiza Bunge)为我国传统中药, 其活性成分的药理作用广泛, 尤其在防治心血管病药物中具有重要作用。丹参水溶性酚酸类物质合成途径由苯丙烷代谢和酪氨酸代谢共同参与而成。转录因子对植物次生代谢起着十分重要的调节作用。我们前期测序结果显示, SmMYC是一个丹参bHLH类转录因子, 可能参与丹参酚酸类化合物生物合成的调控。本研究利用拟南芥miRNA319前体为模板骨架, 通过over-lapping PCR技术构建旨在能对SmMYC进行特异性沉默的artifical miRNA (amiRNA)植物表达载体, 命名为pCambia1302-amiR-SmMYC, 并通过农杆菌介导的遗传转化方法将其导入丹参。Real-time PCR结果显示, 所得转化阳性株系中SmMYC的mRNA水平均呈现不同程度的下降, 同时酚酸类代谢途径中相关酶基因的表达也表现为相应程度的下调; 福林酚法检测结果显示, 转化株系中总酚酸含量均低于同期的野生型丹参。以上实验结果初步显示丹参SmMYC可能作为一个重要的转录因子参与酚酸类活性物质的代谢调控, 同时为进一步研究SmMYC在丹参酚酸类化合物生物合成中的调控功能奠定了基础。

关键词：丹参; bHLH转录因子; amiRNA; 遗传转化

收稿：2013-08-01   修定：2013-09-06

资助：国家自然科学基金(31170281)、陕西省自然科学基金(2011K-16-02-01)、陕西省教育厅自然科学专项基金(11JK0610)和安康学院高层次人才科研启动基金(AYQDZR200926)。

Construction of Artificial microRNA Expression Vector Directing Specific Silencing of the Transcription Factor Gene SmMYC and Subsequent Transformation into Salvia miltiorrhiza

WANG Hao-Ru¹, WANG Jian^1,2, WANG Shi-Ying¹, WANG Zhe-Zhi^1,*

¹Key Laboratory of Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, China; ²College of Agriculture and Life Science, Ankang University, Ankang, Shaanxi 725000, China

Corresponding author: WANG Zhe-Zhi; E-mail: zzwang@snnu.edu.cn

Abstract:

Salvia miltiorrhiza Bunge is the traditional Chinese medicine, whose active constituents have extensive pharmacological activities in the clinical treatment, especially play an important role in heart vascular disease drug. The biosynthesis pathway of phenolic acid, the category of water-soluble active components of Salvia miltiorrhiza, is composed of both phenylpropanoid pathway and tyrosine metabolic one. Transcription factors play a very important role in regulation of the plant secondary metabolism. We speculated that SmMYC, a bHLH transcription factor of Salvia miltiorrhiza, was potentially involved in regulation of the phenolic acid compounds biosynthesis of Salvia miltiorrhiza. In this study, based on the Arabidopsis thaliana miRNA319 precursor, we constructed an artificial miRNA (amiRNA) plant expression vector using the overlap PCR technology, designated as pCambia1302-amiR-SmMYC, which was transformed into Salvia miltiorrhiza, subsequently. Real-time PCR results showed that, in the positive transgenic plants, SmMYC mRNA abundance presented the significantly lower compared with control. The expression level of the enzyme genes involved in phenolic acid compounds biosynthesis pathways rendered lower to some different extents compared with control. Furthermore, the Folin-Ciocalteu assay results showed that the total phenolic acid content in the positive transgenics were much lower than that of wild type. The results above suggested that the SmMYC might be an important transcription factor governing phenolic acids metabolism, and lay a firm foundation for further exploration of SmMYC role in phenolic acid compounds biosynthesis in Salvia miltiorrhiza.

Key words: Salvia miltiorrhiza Bunge; bHLH transcription factor; amiRNA; transformation