青花菜抗坏血酸过氧化物酶基因BoAPX的克隆与表达分析

根据NCBI基因数据库提供的抗坏血酸过氧化物酶(Ascorbate Peroxidase,APX)基因序列设计简并引物,以青花菜(Brassica oleracea var.italica)叶片cDNA和DNA为材料进行PCR扩增,基因定名为BoAPX(Gen-Bank登录号:HQ871864).BoAPX的基因组全长为1 394bp,具7个内含子,编码区全长为753bp,编码250个氨基酸残基.序列分析结果表明,BoAPX的N端中没有信号肽序列,为胞质型APX,BoAPX与已知APX具有较高的同源性.RT-PCR结果表明,BoAPX的表达受霜霉菌(Hyaloperonospora parasitica)诱导,在96h时达最大值,说明BoAPX与霜霉菌抗性相关.以pBI121为植物表达载体,BoAPX为目的基因,成功构建了重组表达载体pBI121-BoAPX.

Cloning and expression analysis of ascorbate peroxidase gene BoAPX from Brassica oleracea var.italica

Degenerate primer pairs were designed according to ascorbate peroxidase gene sequences published in NCBI GenBank database.A gene,designated BoAPX(GenBank accession No.HQ871864) was isolated successfully from leaf cDNA and DNA of Brassica oleracea var.italica,respectively.The full length genomic DNA of BoAPX was 1 394 bp with 7 introns and an open reading frame of 753 bp encoding 250 amino acid residues.Sequence analysis results indicated that BoAPX was a cytosolic one for lacking of signal peptides in N-terminal,and had high homology to known APXs.RT-PCR results revealed that the expression of BoAPX was induced by Hyaloperonospora parasitica,and reached the highest level after 96 h incublation,implying a relationship between BoAPX and broccoli downy mildew.Recombinant vector,named pBI121-BoAPX,was correctly constructed using pBI121 as the expression vector and BoAPX as targe gene.