LmMYB15转录因子调控灰毡毛忍冬绿原酸生物合成的分子机制

Lonicera macranthoides Hand.-Mazz is an important medicinal herb widely distributed and characteristic economic plant in southern China. Chlorogenic acid (CGA) is the major biologically active ingredients and secondary metabolites in L. macranthoides. Improving the content of the CGA by genetic strategies has the attractive application prospect. Evidents showed that MYB is the key regulated transcription factor in the biosynthetic pathway for generating CGA, but there exsit the different regulatory mechanism based on various metabolic pathways in different species. However, there was no report related to the function of MYB transcription factor in L. macranthoides. In our previous work, a R2R3-MYB transcription factor was isolated and designated as LmMYB15. The results of function characterization in transgenic tobacco showed that LmMYB15 might be potentially involved in the biosynthesis of CGA. On the basis of previous work, this project intends to investigate genes function through constructing the sense and RNAi vector and transferring into L. macranthoides callus, thereby the transcriptional regulatory function of LmMYB15 on CGA biosynthesis will be verified and the key CGA biosynthetic enzyme genes will be screened. In addition, yeast-one hybrid (Y1H) and dual-luciferase reporter system will be used to figure out the impacts of LmMYB15 on downstream enzyme genes and confirm its target genes. Furthermore, yeast-two hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays will be employed to isolate and identify the interacting proteins of LmMYB15 and investigate the regulatory effects of interacting proteins on CGA biosynthesis via transgenic technology. The results of this study will provide the theory basis and gene resource for improving the content of CGA by genetic engineering technology in L. macranthoides.

灰毡毛忍冬是我国西南地区大宗道地中药材和特色经济植物，绿原酸是其重要次生代谢产物和核心药效成分，通过遗传改良提高其含量具有诱人的应用前景。证据表明，MYB转录因子参与调控绿原酸合成，但不同物种间因其代谢途径多样而存在不同的调控机制，目前尚未见灰毡毛忍冬MYB转录因子调控绿原酸合成的分子机制研究。申请人前期筛选到一个R2R3-MYB转录因子LmMYB15，转化烟草功能验证表明它可能在绿原酸生物合成中起着重要的调控作用。在此基础上，本研究拟通过超表达和RNAi在灰毡毛忍冬愈伤组织中开展基因功能研究，验证LmMYB15对绿原酸合成的转录调控作用；运用酵母单杂交技术和双荧光素酶报告系统筛选LmMYB15调控的靶基因；运用酵母双杂交和BiFC技术分离鉴定LmMYB15的互作蛋白，通过转基因验证其调控绿原酸合成的功能。研究结果可为利用基因工程技术提高灰毡毛忍冬绿原酸含量提供理论基础和基因资源。

MYB 转录因子在绿原酸代谢中起了重要的调控作用， 但目前尚未见灰毡毛忍冬转录因子调控绿原酸生物合成的研究报道。本项目利用转录组和代谢组分析筛选了4个调控绿原酸合成的候选MYB基因。超表达LmMYB15后转基因植株绿原酸含量显著提高；全长和截短蛋白的转录激活分析表明LmMYB15具有转录激活活性且C端 189-248氨基酸为其转录激活域；利用DAP-Seq 技术鉴定出 MYB15 结合的保守元件ACCTA及 60 个下游靶基因，包括多个转录因子如MYB3、MYB4、KNAT6，激素信号转导元件如IAA26、ETR2，次生代谢相关基因如4CL2，衰老相关基因RD21A、SAG29等； Y1H和DLR实验表明： MYB15能够在体内结合并激活4CL、MYB3、MYB4的启动子，增强这些基因的转录活性，进而调控绿原酸合成；成功构建了灰毡毛忍冬核体系酵母文库，利用Y2H初步筛选了与LmMYB15互作的蛋白，如HSP、HQT等；利用Y1H筛选了绿原酸合成关键酶基因LmHQT3的上游基因，如LIM转录因子、CDPK等。此外，还鉴定了调控绿原酸合成的其它MYB转录因子，如LmMYB12、LmMYB5，对其生物学功能及分子机制做了初步研究，获得了LmMYB12的互作蛋白及LmMYB5的靶基因，具体调控机制还在进行。综上，项目系统阐明了MYB转录因子在灰毡毛忍冬绿原酸等次生代谢物生物合成中的分子机制及调控网络，为揭示绿原酸代谢的转录调控机制奠定基础，也为利用基因工程技术提高绿原酸含量、改良品种提供基因资源。项目执行期间，发表SCI论文3篇，中文期刊1篇，尚有4篇文章正在投稿中；获国家一级行业协会奖励2项；申请国家发明专利2项。培养学术骨干3名，研究生2名，其中课题负责人获重庆英才·创新领军人才等省级人才称号。

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