Cloning and Expression of Squalene Monooxygenase Genes of Dendrobium officinale
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摘要:
目的 克隆铁皮石斛（Dendrobium officinale）甾醇类化合物合成关键酶鲨烯单加氧酶（Squalene Monooxygenase，SQE）基因，并对其进行生物信息学分析和不同营养生长期茎和叶中的表达模式分析。 方法 根据铁皮石斛转录组测序获得带5’末端的SQE基因片段，设计DoSQE1与DoSQE2基因的3’RACE引物，克隆全长cDNA，利用生物信息学分析软件对DoSQE1与DoSQE2基因及其编码蛋白序列进行分析。运用实时荧光定量PCR检测DoSQE1与DoSQE2基因在铁皮石斛营养生长期的8月、10月、12月茎和叶中的表达模式。 结果 DoSQE1基因cDNA序列全长1 796 bp（GenBank 登录号MT160182），含有1个1 554 bp的ORF，编码517个氨基酸；DoSQE2基因cDNA序列全长1 963 bp（GenBank 登录号MT160183），含有1个1 578 bp的ORF，编码525个氨基酸。DoSQE1具有2个跨膜区，分别在4～22 aa和55～72 aa；DoSQE2只有在5～23 aa的1个跨膜区。DoSQE1蛋白在204～476 aa、DoSQE2蛋白在211～484 aa处含有鲨烯环氧酶结构域。系统进化分析表明，DoSQE1与姬蝴蝶兰SQE（XP_020599860.1）的亲缘关系最近，DoSQE2与姬蝴蝶兰SQE（XP_020579136.1）的亲缘关系最近。qRT-PCR检测结果表明，茎和叶中都能检测到2个基因的表达，叶的表达量显著高于茎。DoSQE1基因在8月份表达量最高，DoSQE2基因在10月份表达量最高。 结论 本研究克隆得到DoSQE1和DoSQE2基因，发现DoSQE1和DoSQE2基因的表达模式存在差异，该结果为进一步研究铁皮石斛甾醇类化合物生物合成机理及代谢调控奠定基础。 Abstract:Objective The SQE genes of Dendrobium officinale associated with the key enzyme involving in the sterol biosynthesis, squalene monooxygenase, were cloned for bioinformatics analysis and determination of their expressions in the stems and leaves of the orchid plant at different growth stages. Method The 3'RACE primers of DoSQE1 and DoSQE2 were designed based on SQE fragment with the 5' terminal from the transcriptome data of D. officinale. The full lengths cDNAs of DoSQE1 and DoSQE2 were cloned and a bioinformatics analysis carried out. Expressions of the genes in the stems and leaves in August, October and December were detected by qRT-PCR. Result The full-length of DoSQE1 was 1 796bp (GenBank accession MT160182) containing an 1 554 bp ORF encoding 517 amino acids (aa) and that of DoSQE2 1 963 bp (GenBank accession MT160183) containing an 1 578 bp ORF encoding 525 aa. DoSQE1 had two transmembrane regions at 4–22 aa and 55–72 aa, while DoSQE2 had only one transmembrane region at 5–23 aa. DoSQE1 contained a squalene epoxidase domain at 204–476 aa, and DoSQE2 at 211–484 aa. The phylogenetic analysis showed DoSQE1 to be closely related to SQE of Phalaenopsis equestris (XP_020599860.1), and DoSQE2 to that of Phalaenopsis equestris (XP_020579136.1). Their gene expressions were detected in the stems as well as the leaves by qRT-PCR with the expression in the leaves significantly higher than that in the stems. And, the expression of DoSQE1 peaked in August, whereas, that of DoSQE2 in October. Conclusion DoSQE1 and DoSQE2 were successfully cloned for the study that showed differences in their expressions. The information obtained would lead to further investigation on the biosynthesis mechanism and metabolic regulation of sterols in D. officinale. -
Key words:
- Dendrobium officinale /
- squalene monooxygenase /
- bioinformatics analysis /
- qRT-PCR
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图 1 PCR产物电泳结果
注：M. 分子量标记；A. DoSQE1基因3′RACE；B. DoSQE1基因开放阅读框；C. DoSQE2基因3′RACE；D. DoSQE2基因开放阅读框。
Figure 1. PCR products by electrophoresis
Note: M. DNA marker; A. 3′RACE of DoSQE1; B. ORF of DoSQE1; C. 3′RACE of DoSQE2; D. ORF of DoSQE2.
图 2 DoSQE1与DoSQE2蛋白跨膜区预测
Figure 2. Predicted transmembrane regions in DoSQE1 and DoSQE2
图 3 铁皮石斛DoSQE1和DoSQE2蛋白的二级结构预测
注：大写字母：氨基酸序列；小写字母：二级结构；c：无规则卷曲；h：α螺旋；e：β折叠。
Figure 3. Predicted secondary structures of DoSQE1 and DoSQE2 from D. officinale
Note:capital letters show amino acid sequence; lowercase letters show amino acid chain structure, as c for random coil, h for α-helix, and e for β-extended form.
图 4 预测的铁皮石斛DoSQE1和DoSQE2蛋白三维结构
Figure 4. Predicted 3D structures of DoSQE1 and DoSQE2 from D. officinale
图 5 铁皮石斛DoSQE蛋白系统进化树分析
注：图中各节点处数字代表可信度。
Figure 5. Phylogenetic tree analysis on DoSQE from D. officinale
Note: the numbers at the node of the figure is credibility.
图 6 DoSQE1和DoSQE2基因在铁皮石斛茎和叶中的相对表达量
Figure 6. Expressions of DoSQE1 and DoSQE2 in stems and leaves of D. officinale
表 1 PCR引物序列信息
Table 1. Sequence of PCR primers
引物名称
Primer name引物序列（5′-3′）
Primer sequence（5′-3′）引物用途
Primer usage3DoSQE1F1 CCTTCCATTGCTAACGGAGAGA DoSQE1基因 3′RACE引物3′RACE primers of DoSQE1 3DoSQE1F2 CCTGGAGCACTTCTAATGGGAG 3DoSQE2F1 CTCTTCCTACACCTGGAGCACT DoSQE2基因3′RACE引物 3′RACE primers of DoSQE2 3DoSQE2F2 TATACCTTGCGTAAGCCCGTTG dT-adaptor CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTT 3′RACE接头引物 Adaptor primers of 3′RACE adaptor CTGATCTAGAGGTACCGGATCC DoSQE1F CGCGGTACCATGATGCTTCTCCAGTACA DoSQE1基因ORF克隆引物 primers of DoSQE1 ORF clone DoSQE1R CGCGTCGACGGAGTTCTCATTGTGTAGG DoSQE2F CGCGGTACCATGGTGATGCCACTTTCGTA DoSQE2基因ORF克隆引物 primers of DoSQE2 ORF clone DoSQE2R CGCGTCGACGTTGGAAGTTCACCCACGAG DoSQE1-F AGGACGCAAACAGCGAGAG DoSQE1基因表达分析引物 Expression analysis primers of DoSQE1 DoSQE1-R CACCAACGATTCTATGAGGC DoSQE2-F CCCCAGATACCGAGTCAG DoSQE2基因表达分析引物 Expression analysis primers of DoSQE2 DoSQE2-R CCCATCAGAAGTGCTCCAG DoACT-F AGGAAGGCGGCTTTGAATC 内参基因 Reference gene DoACT-R CCATGCCAACCATGACACC 注：下划线碱基为酶切位点。
Note：The ud bases were the enzyme site.
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