枸杞抗坏血酸过氧化物酶基因的克隆与表达分析

Molecular cloning and expression patterns of LcAPX from Lycium chinense

为探索青海枸杞抗坏血酸过氧化物酶(APX)基因信息,并对枸杞APX基因进行生物信息学分析以及基因表达研究,通过RT-PCR和RACE方法克隆枸杞APX基因,利用生物信息学软件预测基因结构,采用实时荧光定量PCR方法分析基因表达的变化。结果显示:枸杞APX基因的全长cDNA序列为1 047bp(Genbank No.KX981601),命名为LcAPX基因。该基因含有1个753bp的完整开放阅读框(ORF),编码250个氨基酸,包含亚铁血红素结合位点、底物结合位点和K^+结合位点。枸杞LcAPX与茄科植物的APX蛋白聚为一类,说明两者的亲缘关系较近。LcAPX基因在枸杞的成熟叶中表达量最高,花中表达量最少。其在聚乙二醇(PEG)、氯化钠(NaCl)胁迫下诱导表达,显示LcAPX在枸杞抗干旱和抗盐逆境过程起一定作用。

The objective of this study was to explore a ascorbate peroxidase (APX) gene in Lycium chinense by the methods of Reverse-Transcription PCR and RACE,and deduce the structure of this gene by bioinformatics,and analyze gene expression by real time PCR.The full-length cDNA of L.chinense APX (designated as LcAPX) was 1047bp (Genbank No.KX981601),and contained a complete open reading frame (ORF) of 753 bp,which encoded 250 amino acid residues.Sequence analysis showed that LcAPX protein contains the heme binding site,substrate binding site,and K^+ binding site.Homology analysis indicated that the deduced LcAPX protein was highly homologoud to other APX proteins from different species.LcAPX protein had closer relationship with APXs from Solanaceae plants than from other plants.The expression of LcAPX gene was the strongest in the mature leaves of L.chinense,and the least in flowers.Furthermore,LcAPX transcription level was induced under the treatment of PEG and NaCl stresses,and LcAPX played a role in resistance to drought and salt stress processes.