| 张晓东，刘勇江，沙 漠，赵晶晶，李会平，张 喜.禹白芷苯丙氨酸解氨酶基因 AdPAL1 的克隆与表达分析[J].广东农业科学,2023,50(5):1-10 |
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禹白芷苯丙氨酸解氨酶基因 AdPAL1 的克隆与表达分析 |
| Cloning and Expression Analysis of Phenylalanine Ammonialyase Gene AdPAL1 in Angelica dahurica cv. Yubaizhi |
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| DOI：10.16768/j.issn.1004-874X.2023.05.001 |
| 中文关键词: 禹白芷 欧前胡素 苯丙氨酸解氨酶 组织特异性表达 基因克隆 生物信息学分析 原核表达 |
| 英文关键词: Angelica dahurica cv. Yubaizhi imperatorin phenylalanine ammonia-lyase tissue specific expression gene cloning bioinformatics analysis prokaryotic expression |
| 基金项目:河南省自然科学基金面上项目（232300420027）；河南省人力资源和社会保障厅留学人员科研择优资助项目（2023039）；河南省大学生创新项目（S202110480050） |
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| 中文摘要: |
| 【目的】探究禹白芷中欧前胡素含量与苯丙氨酸解氨酶基因 AdPAL1 表达的相关性，并对 AdPAL1进行克隆和原核表达。【方法】使用高效液相色谱法测定禹白芷快速生长期和收获期根和叶中欧前胡素的含量，并使用实时荧光定量 PCR（qRT-PCR）方法测定 AdPAL1 基因在同时期根和叶中的表达情况，以观察欧前胡素含量与 AdPAL1 基因表达是否具有相关性。此外，以禹白芷转录组为基础，采用逆转录 PCR（RT-PCR）技术从禹白芷根中克隆 AdPAL1 基因，并在大肠杆菌中进行表达。【结果】禹白芷快速生长期根中的欧前胡素含量远高于叶，而 AdPAL1 基因在根中的表达量也远高于叶。与快速生长期相比，收获期根中欧前胡素含量略有下降，AdPAL1 基因在根中的表达量也同时下降。这些结果表明 AdPAL1 基因表达与欧前胡素含量具有相关性。此外，生物信息学分析显示，禹白芷 AdPAL1 基因（GenBank 登录号：OQ822236）开放阅读框长 1 764 bp，编码 557 个氨基酸，其蛋白相对分子质量为 61.10 kD，等电点为 5.92，且具有苯丙氨酸解氨酶保守结构域（IPR005922，
1~547），属于 PAL 蛋白家族成员。AdPAL1 蛋白定位于细胞质，主要由 α- 螺旋和无规则卷曲构成，与石防风属植物 KpPAL2 蛋白亲缘关系最近，序列相似性高达 99.28%。AdPAL1 基因在大肠杆菌中表达的重组蛋白相对分子质量约为 84.00 kD，与预期蛋白大小一致。【结论】初步确定禹白芷根和叶中欧前胡素含量与 AdPAL1 基因表达相关，并成功克隆 AdPAL1 基因，在大肠杆菌中成功表达。上述结果为进一步研究 AdPAL1 基因在欧前胡素生物合成中的作用机制提供参考。 |
| 英文摘要: |
| 【Objective】This study aimed to explore the correlation between the content of imperatorin and the
expression of the phenylalanine ammonia-lyase gene (AdPAL1) of Angelica dahurica cv. Yubaizhi (YBZ). In addition,
AdPAL1 was cloned and expressed in prokaryotes.【Method】The content of imperatorin in the roots and leaves of YBZ
during its rapid growth stage and harvest stages was measured using high-performance liquid chromatography (HPLC), and
the expression of the AdPAL1 gene in the roots and leaves during the same stages was detected using real-time fluorescent
quantitative PCR (qRT-PCR) to observe whether there was a correlation between imperatorin content and the expression of the AdPAL1 gene. Furthermore, the AdPAL1 gene was cloned from the root of YBZ based on its transcriptome using reverse
transcription-polymerase chain reaction (RT-PCR) technology, and it was expressed in Escherichia coli.【Result】During the
rapid growth period of YBZ, the content of imperatorin in the roots is much higher than that in the leaves, and the expression level
of AdPAL1 in the roots is also much higher than that in the leaves. Compared with the rapid growth stage, the content of imperatorin
in the roots slightly decreases during the har vest stag e, and the expression level of AdPAL1 gene in the roots also decreases at the
same time. These results indicate a correlation between the expression of AdPAL1 gene and the content of imperatorin. In addition,
bioinformatics analysis show that the AdPAL1 gene (GenBank accession number: OQ822236) of YBZ has an open reading frame
length of 1 764 bp and encods 557 amino acids. The relative molecular weight of AdPAL1 protein is 61.10 kD, and its isoelectric
point is 5.92. It has a conserved domain of phenylalanine ammonia lyase (IPR005922, 1-547), which belongs to the PAL protein
family and it is located in the cytoplasm and is mainly composed of α- helixes and irregular coils. AdPAL1 has the closest genetic
relationship with the KpPAL2 protein in Kitagawia praeruptora, with a local sequence similarity of up to 99.28%. The relative
molecular weight of the recombinant protein of AdPAL1 gene expressed in E. coli is about 84.00 kD, which is consistent with
the expected protein size.【Conclusion】The correlation between the content of imperatorin in roots and leaves of YBZ and the
expression of AdPAL1 gene was preliminarily determined, and the AdPAL1 gene was cloned and successfully expressed in E. coli.
These results provide a reference for further research on the mechanism of AdPAL1 gene in imperatorin biosynthesis. |
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