选择合适的内参基因是提高实时荧光定量PCR分析(q RT-PCR)准确性的重要条件。18S r RNA基因表达范围广、表达量恒定,常作为内参基因应用于实时荧光定量PCR中。为了获得丝瓜18S r RNA基因,并设计合适的荧光定量PCR内参引物,解决丝瓜实时...选择合适的内参基因是提高实时荧光定量PCR分析(q RT-PCR)准确性的重要条件。18S r RNA基因表达范围广、表达量恒定,常作为内参基因应用于实时荧光定量PCR中。为了获得丝瓜18S r RNA基因,并设计合适的荧光定量PCR内参引物,解决丝瓜实时荧光定量PCR检测中无内参基因的现状,通过PCR和序列测定,首次克隆到了丝瓜的18S r RNA基因序列,其长度为1 862 bp,Gen Bank登录号为KM656452。在此基础上设计1对荧光定量PCR引物,该引物特异性强,扩增效率高,在丝瓜各生长发育阶段及各种非生物胁迫条件下均能稳定表达,适合在丝瓜基因表达研究中作为内参基因。该研究结果可为开展丝瓜重要功能基因的表达模式和调控机制的研究奠定基础。展开更多
AIM To analyze sequences of the nuclear ribosomal RNA small subunit (18S rRNA) gene and the chloroplast mat K gene of crude drug Patchouli ( Pogostemon cablin ) in order to provide molecular evidence for identificatio...AIM To analyze sequences of the nuclear ribosomal RNA small subunit (18S rRNA) gene and the chloroplast mat K gene of crude drug Patchouli ( Pogostemon cablin ) in order to provide molecular evidence for identification of Patchouli drug. METHODS To sequence the entire 18S rRNA gene and partial mat K gene of Patchouli from Guangzhou and its substitute Wrinkled Gianthyssop ( Agastache rugosa ) from S ichuan using PCR direct sequencing and to detect the homology of two gene sequen ces between these two crude drugs. RESULTS The complete 18S rRNA gene sequence is 1 805 bp in length for Patchouli from Guangzhou whereas 1 794 bp for Wrinkled Gianthyssop from Sichuan. The 3′ end sequence of mat K gene is 521 bp (747～ 1 268 nt from upstream of ma t K gene) for these two crude drugs. Based on multiple sequence alignment, it i s found that there are 18 variable sites and 11 aligned gap sites in 18S rRNA se quence, 49 variable sites in 3′ mat K sequence between these two crude drug s. The homology is 98 4% for 18S rRNA and 90 6% for 3′ mat K between two crude drugs, respectively. CONCLUSION DNA sequencing can provide an accurate and reliable tool in the crude drug ident ification of Patchouli and its substitute Wrinkled Gianthyssop.展开更多
文摘选择合适的内参基因是提高实时荧光定量PCR分析(q RT-PCR)准确性的重要条件。18S r RNA基因表达范围广、表达量恒定,常作为内参基因应用于实时荧光定量PCR中。为了获得丝瓜18S r RNA基因,并设计合适的荧光定量PCR内参引物,解决丝瓜实时荧光定量PCR检测中无内参基因的现状,通过PCR和序列测定,首次克隆到了丝瓜的18S r RNA基因序列,其长度为1 862 bp,Gen Bank登录号为KM656452。在此基础上设计1对荧光定量PCR引物,该引物特异性强,扩增效率高,在丝瓜各生长发育阶段及各种非生物胁迫条件下均能稳定表达,适合在丝瓜基因表达研究中作为内参基因。该研究结果可为开展丝瓜重要功能基因的表达模式和调控机制的研究奠定基础。
文摘AIM To analyze sequences of the nuclear ribosomal RNA small subunit (18S rRNA) gene and the chloroplast mat K gene of crude drug Patchouli ( Pogostemon cablin ) in order to provide molecular evidence for identification of Patchouli drug. METHODS To sequence the entire 18S rRNA gene and partial mat K gene of Patchouli from Guangzhou and its substitute Wrinkled Gianthyssop ( Agastache rugosa ) from S ichuan using PCR direct sequencing and to detect the homology of two gene sequen ces between these two crude drugs. RESULTS The complete 18S rRNA gene sequence is 1 805 bp in length for Patchouli from Guangzhou whereas 1 794 bp for Wrinkled Gianthyssop from Sichuan. The 3′ end sequence of mat K gene is 521 bp (747～ 1 268 nt from upstream of ma t K gene) for these two crude drugs. Based on multiple sequence alignment, it i s found that there are 18 variable sites and 11 aligned gap sites in 18S rRNA se quence, 49 variable sites in 3′ mat K sequence between these two crude drug s. The homology is 98 4% for 18S rRNA and 90 6% for 3′ mat K between two crude drugs, respectively. CONCLUSION DNA sequencing can provide an accurate and reliable tool in the crude drug ident ification of Patchouli and its substitute Wrinkled Gianthyssop.
