树莓RiPAL1基因克隆及生物信息学分析

Cloning and Bioinformatics Analysis of RiPAL1 Gene of Raspberry

摘要:

苯丙氨酸解氨酶（Phenylalanine Ammonia Lyase，PAL）是苯丙烷类代谢的关键酶，能调节树莓中黄酮类的生物合成。为了深入研究树莓中RiPAL1基因的功能与调控机制，以树莓果实为材料提取总RNA，合成cDNA第一链，然后成功克隆出树莓果实RiPAL1基因，并对其进行了相关的生物信息学分析。结果表明，RiPAL1基因的编码序列（Coding Sequence，CDS）全长为2 133 bp，编码710个氨基酸，分子量为77.50 ku，等电点为6.21。RiPAL1蛋白疏水性最大值为4.50，最小值为-4.50，平均疏水指数为-0.49，为亲水性蛋白，无跨膜结构，无信号肽。含有4个N糖基化位点和67个特异性激酶位点，二级结构以α螺旋为主，三级结构为同型四聚体。多序列比对显示RiPAL1蛋白具有典型的苯丙氨酸解氨酶保守结构域，系统进化分析表明RiPAL1蛋白与同科植物草莓亲缘关系最近。该研究可为深入研究RiPAL1基因参与树莓果实黄酮类物质生物合成的分子机制提供参考。

Abstract:

Phenylalanine ammonia-lyase (PAL) is a key enzyme in the metabolism of phenylpropane that regulates the biosynthesis of flavonoids in raspberry. To elucidate the function and regulation mechanism of the RiPAL1 gene in raspberry, total RNA was extracted from raspberry fruits to synthesize first-strand cDNA. The gene was successfully cloned, and related bioinformatics analyses were carried out. Our results showed that the coding sequence (CDS) of the RiPAL1 gene was 2 133 bp in length, encoding 710 amino acids, with a molecular weight of 77.5 ku and an isoelectric point of 6.21. The maximum hydrophobicity of RiPAL1 protein was 4.50, its minimum hydrophobicity was -4.50, and its average hydrophobicity index was -0.49. Based on these, it was determined that it is a hydrophilic protein with no transmembrane structure and no signal peptide. The RiPAL1 protein contained 4 N-glycosylation sites and 67 specific kinase sites. Its secondary structure was mainly composed of α-helices, while its tertiary structure was a homotetramer. Multiple sequence alignment showed that the RiPAL1 protein has a typical conserved domain of phenylalanine ammonia-lyase. Phylogenetic analysis indicated that the RiPAL1 protein is closely related to that of strawberry. This study provides a reference for further research on the molecular mechanism of the RiPAL1 gene in the biosynthesis of flavonoids in raspberry fruit.