EuDQD/SDH6基因在巨尾桉莽草酸途径中的功能分析

Functional Analysis of Shikimate Dehydrogenases Genes EuDQD/SDH6 in Eucalyptus grandis×E.urophylla

莽草酸脱氢酶家族是多酚类物质合成的关键分支酶,本研究体外分离获得巨尾桉EuDQD/SDH6基因(KY401152.1),在大肠杆菌中表达并纯化了DQD/SDH6蛋白,以莽草酸为底物的DQD/SDH6酶动力学参数为Km=29.93 mmol/L,Vmax=(149.96±0.003) mmol·L^(-1)·s^(-1),表明该蛋白具有SDH活性。实时定量PCR技术研究Eu DQD/SDH6在巨尾桉中的表达特性,发现Eu DQD/SDH6主要在叶片中表达,其次是茎部,根部最少,随着叶片的成熟Eu DQD/SDH6的表达量也逐渐减少。综合分析了实时定量PCR、SDH酶活性和HPLC法测定的酚酸含量的数据,研究巨尾桉DQD/SDH6的酶功能。Eu DQD/SDH6的基因表达量与SA、PCA累积量均呈负线性相关,与GA累积量正线性相关,但关联性较弱,推测桉树DQD/SDH6在巨尾桉中的生物功能可能是以3脱氢莽草酸为底物合成没食子酸,为单宁合成提供前体。Eu DQD/SDH6基因的表达量与硫酸木质素的含量呈正相关,推测Eu DQD/SDH6基因的表达影响木质素在巨尾桉中的累积。

Shikimate dehydrogenase family is the key branch enzyme of polyphenols synthesis.In this study,EuDQD/SDH6(ky401152.1)was isolated from Eucalyptus grandis xE.urophylla.The DQD/SDH6 protein was expressed and purified in E.coli.The kinetic parameters of DQD/SDH6 with shikimate as substrate were K_(m)=29.93 mmol/L,V_(max)=(149.96±0.003)mmol·L^(-1)· s^(-1),which indicated that the protein had SDH activity.Real time quantitative PCR was used to study the expression characteristics of EuDQD/SDH6 in Eucalyptus.It was found that EuDQD/SDH6 was mainly expressed in leaves,followed by stems and roots.With the maturity of leaves,the expression of EuDQD/SDH6 gradually decreased.The data of real-time quantitative PCR,SDH enzyme activity and phenolic acid content determined by HPLC were statistically analyzed to study the enzyme function of DQD/SDH6.The expression of EuDQD/SDH6 was negatively correlated with the accumulation of SA and PCA,andpositively correlated with the accumulation of GA.It is speculated that the biological function of DQD/SDH6 in Eucalyptus may synthesize gallic acid with 3-dehydroashikimic acid as the substrate and provide precursors for tannin biosynthesis.The expression of EuDQD/SDH6 may affect the accumulation of lignin in Eucalyptus grandis x E.urophylla because it was positively correlated with the Kalson lignin content in the transgenic Eucalyptus.