Objective Shikimate dehydrogenase (SDH) and dehydroquinic dehydratase (DQD) in plant shikimate pathway form dimer complex enzyme (DQD/SDH), which is the direct or indirect precursor of lignin, tannin and flavonoids biosynthesis. In the study, three EuDQD/SDH5 genes (Accession No.: KY401151.1, KY401150.1 and KY401149.1) were isolated from Eucalyptus grandis × E. urophylla. The study of enzyme kinetics and expression characteristics of the three enzymes in E. grandis was an important basis for the study of shikimate dehydrogenase family in Eucalyptus.
Method EuDQD/SDH5 was cloned by RT-PCR and expressed in E. coli. The purified protein and enzyme activity were analyzed, and the expression characteristics of EuDQD/SDH5 genes in different tissues of E. grandis were studied by real-time quantitative PCR.
Result The prokaryotic expression system of three mutant genes of EuDQD/SDH5 was constructed successfully. All the three DQD/SDH5 proteins had SDH activity, but their affinity for shikimic acid was different due to the difference of gene sequences. In plants, EuDQD/SDH5-10 had the highest catalytic efficiency for shikimic acid, while EuDQD/SDH5-6 had the lowest catalytic efficiency for shikimic acid. In Eucalyptus tissue culture seedlings, the expression of EuDQD/SDH5 gene was the highest in leaves, slightly lower in stems, and the lowest in roots. The expression of EuDQD/SDH5 was the highest in the leaves of the first internode, and it was similar and low in the leaves of the middle internode and the lowest internode. The expression of EuDQD/SDH5 gene in transgenic Eucalyptus grandis × E.urophylla was also decreased while the content of lignin was decreased.
Conclusion The results showed that EuDQD/SDH5 had SDH activity with shikimic acid as substrate. The gene was differentially expressed mainly in young leaves and stems. It may provide precursor for lignin biosynthesis of Eucalyptus grandis × E. urophylla.
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