红景天甙生物合成依赖的酪氨酸代谢支路途径研究

Salidroside is a representative active ingredients in Rhodiola plants. The previous studies shown that salidroside was derived from tyrosine, and the compound 4-Hydroxyphenylacetaldehyde (4-HPAA) as a precursor for salidroside biosynthesis was derived from different tyrosine metabolism pathways. However, it is still unclear what metabolic pathway dominate the biosynthesis of 4-HPAA from tyrosine. In Rhodiola crenulata plants, two metabolic pathways may involve in tyrosine converting to 4-HPAA, including tyrosine amino transfer pathway catalyzed by TAT and aromatic aldehyde synthesis pathway catalyzed by AAS. The role of the two tyrosine metabolic pathways need to be parsed for the biosynthesis of salidroside. In order to seek the dominant/correct pathway involved in salidroside biosynthesis, the plant materials R. crenulata, a traditional herb in Tibet, will be used in this study. Base on the genomic information of R. crenulata，we will clone and identification these genes coding TAT and AAS. The relation between the genes express space-time specificity and the accumulation of salidroside will be investigated by qPCR and HPLC. Subsequently, the biologic activity of TAT and AAS will be tested, and the related dynamics properties will be assayed. Additionally, the content of salidroside will be analyzed in the transgenic hairy roots with overexpression or silence of TAT/ASS. These results will explain that the effect of TAT and AAS on salidroside biosynthesis, respectively.

红景天甙是红景天代表性有效成分。现有研究证明红景天甙起源于酪氨酸，酪氨酸经过不同途径代谢，生成4-HPAA作为红景天甙的前体。然而从酪氨酸代谢生成红景天苷前体4-HPPA的生物合成途径尚不清楚。在大花红景天中，酪氨酸可能通过2个不同途径生成4-HPAA，包括（1）酪氨酸氨基转运酶（TAT）催化途径；（2）芳香醛合成酶（AAS）催化途径。这两条酪氨酸代谢途径在红景天甙生物合成中所起具体作用有待解析。为寻找红景天甙主导/正确的生源途径，本研究以藏药材大花红景天为材料，在我们自己测定的大花红景天全基因组基础上，克隆鉴定TAT和AAS基因，考察其表达时空特异性和红景天甙积累关系，研究TAT和AAS蛋白质活性和相关动力学性质，采用转基因技术过表达/降低TAT和AAS在大花红景天中表达，进一步研究其对红景天甙生物合成的影响，从而阐明TAT和AAS各自主导的酪氨酸代谢途径在红景天甙生物合成中的作用。

红景天甙是珍稀濒危藏药材大花红景天的代表性有效成分，具有抗氧化化、延缓衰老、抑制肿瘤等多种生物学功能，是医药、化妆品和食品添加剂的重要原料。解析红景天甙代谢途径是利用代谢工程技术培育红景天甙高产品种的前提。本研究完成了大花红景天全基因测序、组装和分析，鉴定了Rc4HPAAS、RcTAT和RcUGT1基因；Rc4HPAAS催化酪氨酸生成4-HPAA从而参与红景天甙合成，RcTAT催化酪氨酸生成4-HPP从而对红景天甙生物合成途径形成竞争，RcUGT1催化酪醇生成淫羊藿次苷D2。随后采用不同的代谢工程策略分别利用Rc4HPAAS与RcTAT提高了红景天甙的含量。Rc4HPAAS过表达株系中红景天甙含量为对照株系的1.31-3.26倍，RcTAT干扰株系中红景天甙含量为对照株系的6-60倍。通过本项目研究，不仅发现了Rc4HPAAS在红景天甙生物合成途径的关键作用，还发现RcTAT介导的红景天甙生物合成竞争支路，为采用代谢工程技术提高红景天甙生物合成提供了科学依据；基于上述发现采用代谢工程技术实现了红景天甙的高产，具有良好应用前景。