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目的:建立金铁锁β-actin基因实时荧光定量PCR方法,为金铁锁实时荧光定量PCR的检测提供了内参基因。方法:根据Gen Bank上β-actin基因保守区域设计特异性引物,利用PCR扩增的方法扩增β-actin目的片段。并以β-actin作为内参基因,利用荧光定量PCR技术对糖基转移酶基因(UGT)在金铁锁根、茎、叶组织中的表达情况进行分析。结果:扩增到金铁锁的β-actin基因序列,长度为153 bp,与王不留行、鹅肠菜、马齿苋的β-actin有较高的同源性。而糖基转移酶基因在以β-actin作为内参基因时能够在金铁锁的不同组织稳定表达。结论:金铁锁β-actin基因是一个可靠的内参基因,适合在金铁锁三萜皂苷生物合成相关功能基因的表达研究中作为内参基因。
OBJECTIVE: To establish a real-time fluorescence quantitative PCR method for β-actin gene of gold lock and provide a reference gene for detection of gold lock real-time fluorescence quantitative PCR. Methods: According to the conserved region of β-actin gene on GenBank, specific primers were designed and the target fragment of β-actin was amplified by PCR. The expression of glycosyltransferase gene (UGT) in the roots, stems and leaves of A. tanguticus was analyzed by fluorescence quantitative PCR using β-actin as an internal reference gene. Results: The sequence of β-actin gene amplified to Caragana trifoliatus was 153 bp in length, which was highly homologous to β-actin gene of Kingfisher, Chickweed and Purslane. The glycosyltransferase gene in β-actin as a reference gene can be stably expressed in different tissues of the gold lock. CONCLUSION: The β-actin gene is a reliable internal control gene and suitable as a reference gene in the study of the expression of functional genes involved in the biosynthesis of triterpene saponins.