芋头过氧化物酶基因克隆

Cloning of peroxidase gene in taro

根据芋头的密码子使用频率,用i CODEHOP设计简并引物扩增芋头过氧化物酶基因序列,待芋头过氧化物酶基因非侧翼序列扩增成功后,依据其序列合成hiTAIL-PCR引物扩增芋头过氧化物酶基因的两侧序列。结果从芋头基因组DNA中克隆出1 165 bp序列,经同源性分析为过氧化物酶基因的部分序列;再利用hiTAIL-PCR技术可以扩增出芋头过氧化物酶基因的两侧序列,扩增序列为689 bp,测序结果能与1 165 bp序列拼接到一起,长1 854 bp。与已知的过氧化物酶进行序列比对,发现两侧还有氨基酸的编码序列,因此进行第2次hiTAIL-PCR,扩增出165 bp芋头过氧化物酶基因的侧翼序列,拼接后得到2 019bp。用Softberry上的基因预测软件分析后,发现包含过氧化物酶4个外显子,同源性分析表明,此过氧化物酶基因为含血红素的第三类过氧化物酶。

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.