| 杨晓丽，罗桂花，陈志，乔枫.独一味基因表达SYBR Green I 荧光定量
PCR检测方法的建立[J].广东农业科学,2014,41(12):133-137 |
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独一味基因表达SYBR Green I 荧光定量
PCR检测方法的建立 |
| Establishment of a real-time PCR assay based onSYBR green I for detection of the gene expressionof Lamiophlomis rotate (Benth.) |
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| DOI： |
| 中文关键词: 独一味 SYBR Green I 荧光定量RT-PCR CHS PAL |
| 英文关键词: Lamiophlomis rotate (Benth.) SYBR green I real-time PCR CHS PAL |
| 基金项目:教育部春晖计划项目(Z2010076） |
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| 摘要点击次数: 1398 |
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| 中文摘要: |
| 为了建立一种检测独一味mRNA 表达水平的SYBR Green I 荧光定量Real-Time PCR 方法。根据独一
味查尔酮合成酶(LrCHS）和苯丙氨酸解氨酶(LrPAL）基因序列设计特异性引物，运用Real-Time RT-PCR 方法建立
独一味基因的荧光定量标准曲线，进一步研究独一味CHS 和PAL 基因在不同组织中的表达。结果显示，扩增曲线在
1伊105耀1伊1011 copies/滋L 范围内有很好的线性关系，扩增相关系数在0.995 以上。熔解曲线分析表明，产物为特异单
峰，无引物二聚体，具有较高的特异性和灵敏度。独一味CHS 基因在不同组织中表达量顺序为花>叶>叶柄>根>茎，
PAL 基因表达量顺序为叶柄>叶>花>根>茎。建立的检测方法能够成功用于独一味CHS 和PAL 基因表达的检测，为
研究独一味在mRNA 水平的定量分析提供技术平台。 |
| 英文摘要: |
| This study developed a real-time reverse-transcription polymerase chain reaction (RT-PCR) method based
on SYBR Green I fluorescent for detection of Lamiophlomis rotate (Benth.) gene mRNA. According to the gene sequence in
the conservative regions of chalcone synthase (LrCHS) and phenylalanine ammonia-lyase (LrPAL) in L. rotate, the specific
primers were designed with LrCHS and LrPAL to construct real-time RT-PCR assay The standard curves showed good
linear relationships with the correlation cofficient abave 0.995 in the range of 1伊105 to 1伊1011 copies/L The melting curve
analysis showed that the product was specific to a single peak and no primer-dimers with high specificity and sensitivity
The expression level of CHS gene in different organization of L. rotate showed flowers>leaves>petioles>roots>stems trend,
and the expression level of PAL gene showed petioles>leaves>flowers>roots>stems trend. This assay could successfully
provide a technical platform to research L. rotate genes at the mRNA level in the quantitative analysis. |
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