关键词: 马铃薯, α-淀粉酶基因, 克隆, 生物信息学

Abstract:

A cDNA for potato α-amylase was amplified by RT-PCR and then cloned. The sequence analysis showed that the cDNA had a 1224bp-ORF, which encodes an α-amylase with 407 amino acid residues (GenBank accession number: GQ406048.1). Semi-quantitative RT-PCR assay was used to detect the expression of the α-amylase genes in potato leaves and stems, and the results showed that the expression in stems was a little stronger than that in leaves. The amino acid sequence was bioinformatically analyzed, including its codon usage bias, physical and chemical properties, subcellular localization and conserved structures. Totally 29 α-amylase genes from the same or different species were taken from the GenBank for constructing a phylogenetic tree. The bioinformatical analyses showed that the putative protein shared 98% identity with a published potato α-amylase (M79328.1) at the amino acid level. The deducted α-amylase also contained a catalytic domain (PF00128 and SM00624) between sites 20 and 348 and a C-terminal beta-sheet domain between sites 349 and 407, which were similar to their counterparts in the amylase family 13. The postulated eight-stranded alpha/beta barrel was also found in the enzyme, which is thought as an active site of α-amylase. According to the constructed phylogenetic tree, the two genes from potato presented closer homology to those from cassava and apple, followed by kidney bean, but distant homology to those from the monocotyledonous plants including barley, rice and maize.

Key words: potato, α-amylase, cloning, bioinformatic analysis