红花菜豆黄烷酮-3-羟化酶基因电子克隆及序列分析

In Silico Cloning and Sequence Analysis of F3H Gene in Phaseolus coccineus

摘要:

黄烷酮-3-羟化酶（F3H）是植物类黄酮物质生物合成途径中的一个关键酶。本文利用电子克隆的方法，采用已经报道的大豆F3H基因cDNA序列为种子序列，搜索红花菜豆EST数据库，获得了假定的红花菜豆F3H基因cDNA的序列。根据假定的cDNA序列设计引物，采用RT-PCR的方法获得了红花菜豆F3H基因cDNA的编码序列。并采用生物信息工具对红花菜豆F3H的性质包括氨基酸序列组成、物理和化学性质、二级和三级结构的特点进行了研究。结果表明，红花菜豆F3H基因cDNA包含一个1128 bp的完整的开放读码框，其编码蛋白由375个氨基酸组成。与大豆黄烷酮-3-羟化酶同源性为92%。其二级结构含有40.8% α螺旋和39.73%无规则卷曲，β折叠较少为4.53%。采用同源模建的方式分析其三维立体结构，表明其三维结构是一个紧密的球状结构。

Abstract:

Flavanone 3-hydroxylase is a key enzyme in the biosynthetic pathway of plant flavonoids. The putative cDNA sequence of Phaseolus coccineus F3H gene was in silico cloned by using Glycine max F3H gene cDNA sequence (AAU06216.1) as a probe. According to the putative cDNA, the primers were designed for RT-PCR. The coding sequence of F3H gene in Phaseolus coccineus cDNA was cloned using RT-PCR. Then the secondary structure and advanced structure of F3H in Phaseolus coccineus were analyzed by bioinformatics methods. The results showed that the cDNA contains an open reading frame of 1128 bp, which encoded the protein containing 375 amino acids. The protein coded by Phaseolus coccineus F3H gene cDNA showed 92% similarity to Glycine max. Its secondary structure contained 40.8% α-helix, 14.93% extended strand, 39.73% random coil and 4.53% β-turn. The homology analysis of three-dimensional structure showed that the three-dimensional structure of the protein was a compact globular structure.