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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.
Degenerate primers were designed according to the Ascorbate Peroxidase (APX) gene sequence provided by the NCBI gene database. The cDNA and DNA of Brassica oleracea var.italica were used for PCR amplification. The gene was named BoAPX ( GenBank accession number: HQ871864) .BoAPX genome has a total length of 1 394 bp with 7 introns and encodes a 753 bp full length coding for 250 amino acid residues.Sequence analysis showed that there was no NAP in BoAPX The signal peptide sequence was cytoplasmic APX and BoAPX had high homology with the known APX.RT-PCR results showed that the expression of BoAPX was induced by Hyaloperonospora parasitica and reached the maximum at 96h, indicating that BoAPX Which is related to the resistance of downy mildew.The recombinant expression vector pBI121-BoAPX was successfully constructed with pBI121 as plant expression vector and BoAPX as the target gene.