苜蓿乙醛脱氢酶基因实时荧光定量PCR检测方法的建立
根据苜蓿基因组ALDH基因和稳定表达Actin基因mRNA序列,设计3对引物.提取苜蓿总RNA,采用oligo dT18作为反转录引物,经反转录获得cDNA,通过优化反应体系及反应条件,建立以苜蓿Aatin基因为内参对照,检测苜蓿乙醛脱氢酶（aldehyde dehydrogenase,ALDH）基因（a亚基和b亚基）的SYBR Green Ⅰ荧光定量PCR方法,苜蓿ALDH基因（a亚基和b亚基）和Actin基因扩增曲线和标准曲线显示其基因扩增效率均大于99.9％,熔解曲线分析表明3个基因的扩增产物为特异性单峰,其Tm分别为（86.4±0.2）,（80.6±0.1）和（83.0±0.1）℃.该...
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| Published in | 西南农业学报 Vol. 27; no. 1; pp. 413 - 418 |
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| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
云南农业大学动物科学与技术学院,云南昆明,650201
2014
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1001-4829 |
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| Summary: | 根据苜蓿基因组ALDH基因和稳定表达Actin基因mRNA序列,设计3对引物.提取苜蓿总RNA,采用oligo dT18作为反转录引物,经反转录获得cDNA,通过优化反应体系及反应条件,建立以苜蓿Aatin基因为内参对照,检测苜蓿乙醛脱氢酶（aldehyde dehydrogenase,ALDH）基因（a亚基和b亚基）的SYBR Green Ⅰ荧光定量PCR方法,苜蓿ALDH基因（a亚基和b亚基）和Actin基因扩增曲线和标准曲线显示其基因扩增效率均大于99.9％,熔解曲线分析表明3个基因的扩增产物为特异性单峰,其Tm分别为（86.4±0.2）,（80.6±0.1）和（83.0±0.1）℃.该方法为利用苜蓿Actin基因作为内参基因进行苜蓿ALDH基因的相对定量表达分析提供了方法学基础. |
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| Bibliography: | Alfalfa;Aldehyde dehydrogenase （ALDH） gene;Real-time PCR 51-1213/S According to the alfalfa genome ALDH gene and stable expression of the Actin gene mRNA sequences,three pairs of primers were designed.Extracting alfalfa total RNA and using an oligo dT18 primer as the revere transcription,the alfalfa （aldehyde dehydmgmme） ALDH genes and Actin-based can be identified and detected.The alfalfa ALDH gene（a subunit and b subunit） and Acin Gene SYBR Green Ⅰ Fluorescence quantitative RT-PCR reaction system and reaction condition have been established and optimized.The amplification curve and standard curve of Alfalfa ALDH gene（a subtnit and b subunit） and the Actin gene indicated that amplification effciency was greaer than 99.9 ％.The melting curve analysis showed that three genes amplification products had a specific single peak,and their Tm were respectively （86.4±0.2）,（80.6±0.1） and （83.0±0.1）℃.Using alfalfaActin gene as reference gene could provide a methodological basis for the alfalfa ALDH gene relative quantitat |
| ISSN: | 1001-4829 |