| 王 梅，赵艳玲.巨尾桉 EuDQD/SDH5 基因的克隆与表达分析[J].广东农业科学,2021,48(4):55-61 |
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巨尾桉 EuDQD/SDH5 基因的克隆与表达分析 |
| Cloning and Expression of EuDQD/SDH5 Genes in Eucalyptus grandis × E. urophylla |
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| DOI：10.16768/j.issn.1004-874X.2021.04.008 |
| 中文关键词: 巨尾桉 莽草酸脱氢酶 酶活性 定量 PCR |
| 英文关键词: Eucalyptus grandis × E. uropHylla shikimate dehydrogenase enzyme activity quantitative PCR |
| 基金项目:福建省科技厅引导性（重点）项目（2018N0018） |
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| 中文摘要: |
| 【目的】植物莽草酸途径中的莽草酸脱氢酶（Shikimate dehydrogenase，SDH）与脱氢奎尼酸脱
水酶（Dehydroquinate dehydratase，DQD）形成的二聚体复合酶（DQD/SDH），是木质素、单宁和黄酮类物质
等生物合成的直接或间接前体。体外分离 3 个巨尾桉 EuDQD/SDH5 基因（GenBank 登录号：KY401151.1、
KY401150.1、KY401149.1），研究 3 个酶的酶动力学及其在巨尾桉中的表达特性，是开展桉树莽草酸脱氢酶家
族研究的重要基础。【方法】利用 RT-PCR 方法克隆 EuDQD/SDH5，原核表达 DQD/SDH5 蛋白，进行纯化蛋白
和酶活性分析，通过实时定量 PCR 技术研究 EuDQD/SDH5 基因在桉树各组织中的表达特性。【结果】EuDQD/
SDH5 的 3 个突变基因的原核表达体系构建成功，3 个 DQD/SDH5 蛋白均具有 SDH 活性，但是因为基因序列
存在差异，导致其对莽草酸的亲和力存在差异，在植物体内 EuDQD/SDH5-10 对莽草酸的催化效率最大，而
EuDQD/SDH5-6 对莽草酸的催化效率最低。在桉树组培苗中 EuDQD/SDH5 基因在叶片中表达量最高，在茎中的
表达量稍低，在根中的表达量最低。盆栽苗不同节间的叶片中，该基因的表达量也有差异，在第一节间的叶片
中 EuDQD/SDH5 表达量最高，在中间节间和最下层节间叶片中的表达量相似且都很低。转基因巨尾桉中木质素
含量降低的植株其 EuDQD/SDH5 基因的表达量也降低。【结论】EuDQD/SDH5 具有以莽草酸为底物的 SDH 活性，
该基因在植物的不同组织和不同节间的叶片中差异表达，主要在幼叶和茎中表达量高，可能为巨尾桉木质素的
生物合成提供前体物质。 |
| 英文摘要: |
| 【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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