Cloning of peroxidase gene in taro

The taro peroxidase gene sequence was amplified using degenerate primers which were designed by iCODEHOP according to the taro codon usage frequency. And the peroxidase gene non-flanking sequence was amplified successfully. Then the peroxidase gene flanking sequences were amplified by hiTAIL-PCR primers which were designed on the basis of the non-flanking sequence. As a result，1 165 bp sequences were successfully cloned from taro genomic DNA，and it was affirmed that they were part of the sequences of peroxidase gene by homology analysis. Then by hiTAIL-PCR technology，the peroxidase gene flanking sequences were amplified，and 689 bp sequences were amplified. The Sequencing result could be spliced together with the 1 165 bp sequences. And the amplified taro peroxidase gene sequences spliced together with its flanking sequences were 1 854 bp. Sequence alignment with known peroxidase，we found that there were amino acid coding sequences on both sides of the amplification sequences. So，the hiTAIL-PCR technology was used as the second time，and 165 bp sequences were amplified. Spliced together with the 1 854 bp sequences，the amplified taro peroxidase gene sequences were 2 019 bp. With the gene on softberry prediction software analysis，it was found that the sequences contained 4 peroxidase exons. Homology analysis showed that the peroxidase gene was Class III heme-containing peroxidase.