RT-PCR克隆甜菜硝酸还原酶cDNA全长序列及分析

Cloning and Sequence Analysis of Nitrate Reductase Gene from Beta vulgaris by RT-PCR

根据GenBank中已公布的甜菜(Beta vulgaris)硝酸还原酶(nitrate reductase)基因序列(gb|ABW 05098.1|),设计引物,以50 mmol.L-1KNO3溶液处理的甜菜幼苗为材料,从总RNA中通过RT-PCR分离得到一个硝酸还原酶基因,其cDNA长2 760 bp,包含了完整的基因编码序列,与已公布的硝酸还原酶基因序列相似性达99%。Southern杂交分析表明,硝酸还原酶基因在甜菜基因组中可能以两个拷贝或低拷贝形式存在。根据其编码的氨基酸序列,利用生物信息学预测了其亚细胞定位和蛋白质的三级结构。

A pair of primers was designed according to the sequence of nitrate reductase gene in GenBank （gb ｜ ABW05098.1 ｜ ）. The homolog cDNA with 2 760 base pairs of nitrate reductase gene was obtained by RT-PCR from total RNA using Beta vulgaris treated by 50 mmol· L^-1 KNO3 as material. DNA sequence analysis showed that it contains the entire open reading frames that encode deduced protein with 905 amino acid residues and shows 99% sequence identity to B. vulgaris nitrate reductase gene. Southern hybridization experiments revealed that nitrate reductase gene might be exist as two copy or low copy in the genome of B. vulgaris. According to the amino acid sequences, the subcellular location and structure of the nitrate reductase were predicted.