| 张晓东,刘勇江,沙漠,等.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,(5):- |
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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 Ammonia-lyase 1 Gene AdPAL1 in Angelica dahurica |
| 投稿时间：2023-04-23 修订日期：2023-05-14 |
| DOI： |
| 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 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】探究禹白芷快速生长期和收获期根和叶中欧前胡素含量与AdPAL1基因表达的相关性，克隆禹白芷苯丙氨酸解氨酶基因AdPAL1，并进行原核表达。禹白芷是河南道地药材，其主要药效成分为香豆素类化合物。欧前胡素是白芷中重要的香豆素成份之一，而香豆素的生物合成起源于苯丙素途径，苯丙氨酸解氨酶是该途径的第一个限速酶。【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。同时，以禹白芷转录组为基础，采用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欧前胡素的含量远高于叶，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（登录号：OQ822236）开放阅读框长1 764 bp，编码557个氨基酸；AdPAL1蛋白相对分子质量为61.10 kD，等电点为5.92；具有苯丙氨酸解氨酶保守结构域 （IPR005922苯丙氨酸解氨酶，1-547），属于PAL蛋白家族成员；该蛋白定位于细胞质，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，局部序列相似性高达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】初步确定了禹白芷根和叶中欧前胡素含量与AdPAL1基因表达的相关性，并克隆了AdPAL1基因，且在大肠杆菌中成功表达。这些结果为进一步研究AdPAL1基因在欧前胡素生物合成中的作用机制奠定基础。 |
| 英文摘要: |
| 【Objective】This study aimed to explore the correlation between the content of imperatorin in the roots and leaves of Angelica dahurica cv. Yubaizhi (YBZ) during its rapid growth and harvest stages and the expression of the AdPAL1 gene. The AdPAL1 gene, encoding phenylalanine ammonia-lyase, was also cloned and expressed in prokaryotes. YBZ is a traditional Chinese medicinal herb from Henan Province, China, and its main active ingredient is coumarin compounds. Imperatorin is an important coumarin component of YBZ, and the biosynthesis of coumarins originates from the phenylpropanoid biosynthesis pathway, in which phenylalanine ammonia-lyase is the first rate-limiting enzyme.【Method】The content of imperatorin in the roots and leaves of YBZ during its rapid growth and harvest stages was measured using high-performance liquid chromatography, and the expression of the AdPAL1 gene in the roots and leaves during the same stages was detected using real-time fluorescent quantitative PCR to observe whether there was a correlation between the content of imperatorin and the expression of the AdPAL1 gene. Furthermore, the AdPAL1 gene was cloned from the root of YBZ based on its transcriptome using 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 gene 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 decreased during the harvest stage, and the expression level of AdPAL1 gene in the roots also decreased at the same time; These results indicate a correlation between the expression of AdPAL1 gene and the content of imperatorin. In addition, bioinformatics analysis showed that the AdPAL1 gene (accession number: OQ822236) of YBZ had an open reading frame length of 1764 bp and encoded 557 amino acids; The relative molecular weight of AdPAL1 protein is 61.10 kD, and its isoelectric point is 5.92; Having a conserved domain of phenylalanine ammonia lyase (IPR005922 phenylalanine ammonia lyase, 1-547), belonging to the PAL protein family; This protein is located in the cytoplasm and is mainly composed of α- helixes and irregular coils; It 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 lay the foundation for further studying the mechanism of action of AdPAL1 gene in the biosynthesis of imperatorin. |
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