PgPDR3基因调控人参皂苷跨膜转运与生物合成的功能研究

The PgPDR3 gene involved in ginsenosides transport has first been cloned and its functional expression analysis in Xenopus oocyte and Panax ginseng cells showed PDR3 transporter gene played function in the transport of total ginsenosides. However PDR3 function involved in ginsenoside components-specific transport and ginsenosides biosynthesis is unclear. Based on these results, Yeast cells (ABC transporter genes knockout) harboring PDR3 gene will be used as ginsenosides precursor and its metabolic components feeding test to quantitatively analyze the changes of the ginsenosides using LC-MS/MS. The specific recognition and interaction between ginsenosides and PDR3 transporter will be further identified by means of the surface plasmon resonance (SPR) and the isothermal titration calorimetry (ITC) to clarify the function of PDR3 specific recognition, transport of ginsenoside components. Using transcriptome sequencing and LC-MS metabolome techniques to comparatively analyze the relationship between the expression of transcriptome and changes of the ginsenoside biosynthesis metabolomic pathway in ginseng cell and hairy root with PDR3 overexpression and CRISPR/Cas9 system knock-out, and then the function of PDR3 in regulating ginsenoside biosynthesis will be further elucidated. To further explore the function played in ginsenoside biosynthesis and accumulation by PDR3 and ginsenoside biosynthases coordinated expression, PDR3 overexpression will be combined with the overexpression of DDS that is considered as the key enzyme of dammarane-type ginsenoside biosynthesis and also with CRISPR/Cas9 system knock-out of the key enzyme genes including β-AS and CAS in the branch metabolism pathway of ginsenoside biosynthesis. Finally, this study will provide a theoretical base and a new strategy in improving the dammarane-type ginsenoside yield through the coordinated regulation of transport and biosynthesis metabolism.

我们已率先克隆人参皂苷转运蛋白候选PgPDR3基因，且在人参和爪蟾卵母细胞表达证实其转运人参总皂苷功能。但PDR3表达蛋白对皂苷组分的特异性转运和皂苷生物合成的调控作用及机制尚不清楚。本研究拟建立PDR3超表达的酵母系并使用皂苷组分喂饲，LC-MS定量测定上述酵母细胞中皂苷含量变化，同时应用SPR和ITC法体外鉴定酵母PDR3蛋白与皂苷组分的互作，进而阐释PDR3蛋白特异性转运皂苷的功能；建立PDR3超表达及CRISPR/Cas9系统敲除的人参细胞和发根系，应用功能组学技术比较分析其转录组表达与皂苷生物合成通路代谢组改变的关系，以阐明PDR3对皂苷生物合成的调控作用与机制；再建立PDR3与皂苷生物合成主途径DDS超表达、分支代谢βAS/CAS敲除的人参发根系，从转录组及代谢组水平探究PDR3协同调控在皂苷生物合成与积累中的作用。最终为实现达玛烷型人参皂苷代谢工程生产与利用提供新的理论依据。

为深入解析PgPDR3在人参皂苷转运及其调控人参皂苷生物合成与积累的功能。该研究主要建立PgPDR3超表达和沉默的人参细胞和发根系，结合HPLC/MS代谢组学等技术，探究PgPDR3与生物合成关键酶基因协同控制及转录因子调控在人参皂苷生物合成与积累中的作用及机制。研究取得了如下重要成果：.（1）明确了PgPDR3转运人参皂苷的功能及PgPDR3转运对人参皂苷生物合成代谢流的调控，阐释了PgPDR3与生物合成关键酶基因协同控制及转录因子调控在人参皂苷生物合成与积累中的作用及机制；从人参不定根中分离到一个编码R2R3-MYB蛋白的新基因PgMYB2，其亚细胞定位洋葱表皮细胞核，在人参根中表达量最高，植物激素茉莉酸甲酯（MeJA）对PgMYB2表达有显著的诱导作用。在酵母菌株Y1H-Gold中发现了PgMYB2蛋白与人参皂苷生物合成关键酶Dammarenediol Synthase（DDS）启动子之间的结合相互作用。此外，电泳迁移率转移分析（EMSA）鉴定了这种相互作用的结合位点，人参过表达PgMYB2表明能正调控PgDDS的表达。.（2）基于转录组测序筛选发现二个新的PgMYB3 和PgERF1转录因子，其在人参皂苷生物合成调控中的功能仍在研究中。此外，首次克隆了2个人参细胞色素P450还原酶基因PgCPR1和PgCPR2，均具有还原酶活性，其高于拟南芥AtCPR1活性。且受MeJA诱导后人参CPR基因的表达量增加与诱导后人参皂苷产量的增加一致，为构建合成人参皂苷的酵母工程菌提供了新的催化元件。.该研究为应用PgPDR3与PgDDS和转录因子协同调控人参皂苷生物合成 与积累并提高其产量奠定了重要理论与实验基础。.主要结果部分已在国际SCI期刊共发表6篇（Intl Journal of Molecular Sciences, Acta Biochimica et Biophysica Sinica, Journal of Microbiology and Biotechnology, Molecular Medicine Reports, Molecular Medicine）、中文核心期刊1篇，投稿Nature Communications, J Ginseng Research及 Neuroscience期刊3篇；申请国家发明专利2项及国际专利PCT 1项。