Cloning and expression analysis of Ipomoea batatas (L.) Lam. gene IbPAL
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摘要: 本研究基于甘薯（Ipomoea batatas（L.）Lam.）与绿原酸合成代谢相关的转录组数据克隆了苯丙氨酸解氨酶基因IbPAL，通过蛋白序列比对、系统进化分析、二级和三维蛋白结构预测、跨膜结构域以及启动子分析，对该基因的氨基酸序列和启动子序列的结构特征进行了解析；通过qRT-PCR技术分析该基因在不同甘薯品种的根、茎、茎尖和叶中的表达情况，并对其进行亚细胞定位验证。结果显示，IbPAL的CDS序列全长2130 bp，编码709个氨基酸。其氨基酸序列与已知物种的氨基酸序列同源性在80%以上。IbPAL蛋白以α螺旋和随机卷曲为主，具有1个可能的跨膜螺旋区。该基因启动子中包含多个顺式作用元件。表达模式分析结果表明，IbPAL主要在茎和茎尖中表达，其在甘薯品种‘EC16’4个组织器官中的表达量均显著高于其他品种。亚细胞定位结果显示该基因在细胞膜和细胞核上均可以表达。Abstract: In this research, the phenylalanine ammonia-lyase gene IbPAL was cloned based on transcriptome information related to chlorogenic biosynthesis metabolism in Ipomoea batatas (L.) Lam. Its amino acid (aa) structure was also analyzed by means of protein sequence alignment, phylogenetic analysis, secondary and three-dimensional protein structure prediction, and transmembrane domain and promotor analysis. The expression patterns of IbPAL in roots, stems, meristems, and leaves were further determined by qRT-PCR, with expression locations validated by subcellular experiments. Results showed that the CDS of IbPAL was 2130 bp in length, encoding 709 amino acids. Homologous alignment of the amino acid sequences indicated that IbPAL had high similarity (> 80%) with other reported species. Its protein mainly consisted of α-helixes and random coils and was predicated to have a transmembrane domain. Its promoter contained several cis-elements. IbPAL was mainly expressed in stems and meristems and its expression levels in the four tissues of sweet potato variety ‘EC16’ were significantly higher than that in other varieties and lines. The subcellular experiments indicated that the gene was expressed in the cell nucleus and cytomembrane.
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Keywords:
- Ipomoea batatas /
- IbPAL /
- Clone /
- qRT-PCR /
- Subcellular location
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