丹参SmHPPR1基因调控丹酚酸生物合成的研究

SmHPPR1 from Salvia miltiorrhiza regulated the biosynthesis of salvianolic acids

丹参是临床上治疗心脑血管疾病的常用中药,其主要水溶性活性成分为迷迭香酸和丹酚酸B,由苯丙烷类代谢途径产生。4-羟基苯丙酮酸还原酶(HPPR)是苯丙烷代谢途径中的关键酶。本课题组前期在丹参中克隆了SmHPPR1基因并构建了植物表达载体。在此基础上,获得了携带SmHPPR1的转基因拟南芥阳性植株并测定4-羟基苯乳酸的含量;诱导了过表达SmHPPR1丹参毛状根并进行有效成分含量测定和转录组分析。结果显示:携带SmHPPR1的T1代转基因拟南芥中4-羟基苯乳酸含量为0.594 mg·g^(-1);SmHPPR1-OE-3毛状根中的迷迭香酸、丹酚酸B、总丹酚酸含量分别是control-3毛状根的1.09、1.29、1.15倍,丹参素是control-3毛状根的36.26%;转录组分析显示,SmHPPR1过表达引起了SmTAT2表达量上调;蛋白-蛋白相互作用表明CYT(TR74706_c0_g1)、NADP^(+)(TR26565_c0_g1)和NADP^(+)(TR68771_c0_g1)是网络的中心节点并参与代谢过程、细胞过程等。以上研究表明,SmHPPR1在携带SmHPPR1的转基因拟南芥中能催化4-羟基苯丙酮酸还原为4-羟基苯乳酸;丹参毛状根中过表达SmHPPR1可以通过协同调控其他途径基因来提高丹酚酸的含量。

Salvia miltiorrhiza Bunge is a traditional Chinese medicinal herb widely used to treat cardiovascular and cerebrovascular diseases at clinic.Its main water-soluble components are rosmarinic acid(RA)and salvianolic acid B(SAB),which are produced by phenylpropanoid pathway.4-Hydroxyphenylpyruvate reductase(HPPR)is a key enzyme in phenylpropanoid metabolism pathway.SmHPPR1 was cloned from S.miltiorrhiza and was constructed into plant expression vector pJR-SmHPPR1.On this basis,SmHPPR1 transgenic Arabidopsis plants were induced and the content of 4-hydroxyphenyllactic acid(pHPL)was determined.SmHPPR1-overexpressing(SmHPPR1-OE)hairy roots of S.miltiorrhiza were obtained and the concentration of active components and transcriptome analysis were performed.The results showed that the concentration of pHPL in SmHPPR1 transgenic Arabidopsis T1 was 0.594 mg·g^(-1)dry weight.The concentration of RA,SAB and total salvianolic acid in SmHPPR1-OE-3 hairy roots were 1.09,1.29,1.15 times of that in control-3,respectively,and the content of Danshensu was 36.26%of that in control-3.Transcriptomic analysis revealed that overexpression of SmHPPR1 caused the upregulation of other phenylpropanoid pathway genes like SmTAT2.Protein-protein interaction indicated CYT(TR74706_c0_g1),NADP+(TR26565_c0_g1)and NADP+(TR68771_c0_g1)is the central node of the network and participated in metabolic process and cellular process.The tracking work in this study proved that SmHPPR1 could catalyze the reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid in SmHPPR1 transgenic Arabidopsis,and SmHPPR1-overexpressing in hairy roots of S.miltiorrhiza could increase the concentration of salvianolic acids through syner‐gistically regulating other pathway genes.