莫俊杰,梁钾贤,胡汉桥,黄 红,陈 妤.芋头过氧化物酶基因克隆[J].广东农业科学,2016,43(9):37-43 |
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芋头过氧化物酶基因克隆 |
Cloning of peroxidase gene in taro |
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DOI:10.16968/j.issn.1004-894X.2016.09.006 |
中文关键词: 芋头 过氧化物酶基因 hiTAIL-PCR 克隆 |
英文关键词: taro peroxidase gene hiTAIL-PCR cloning |
基金项目:国家公益性行业(农业)科研专项经费子课题分项目(200903017-08) |
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中文摘要: |
根据芋头的密码子使用频率,用 iCODEHOP 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2 019
bp。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。 |
英文摘要: |
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. |
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