摘要
目的克隆刺五加Eleutherococcus senticosus的肌动蛋白(actin,ACT)基因并使其成为可用的内参基因。方法运用RT-PCR法克隆ACT基因的部分序列,并以其为内参基因进行半定量PCR和real-time PCR分析。结果克隆了长度为1 031bp的刺五加ACT基因,推测其编码343个氨基酸,与光皮桦Betula luminifera、陆地棉Gossypium hirsutum、茶树Camellia sinensis的ACT氨基酸序列同源性分别为99.42%、98.83%、98.54%。刺五加ACT基因在不同器官和不同生长发育时期的表达量基本恒定,以其为内参基因的半定量PCR和real-time PCR均具有良好的扩增效果和重现性。结论首次分离并报道了刺五加ACT的cDNA克隆,证实该序列可以作为基因表达分析的内参基因,建立了其real-time PCR反应体系。
Objective To clone the actin （ACT） gene ofEleutherococcus senticosus, and to make the gene a valuable internal gene. Methods Part of the sequence of ACT gene was cloned by real-time PCR （RT-PCR）, and the sequence was used as internal control gene for analyses of semiquantitative PCR and RT-PCR. Results The ACT gene （1 031 bp） orE. senticosus was cloned, coding 343 amino acids. To compare the amino acid sequence ofE. senticosus ACT gene with those ofBetula luminifera, Gossypium hirsutum, and Camellia sinensis, the amino acid homology was 99.42%, 98.83%, and 98.54%. The expression of ACT in different organs of E. senticosus during various growing periods was constant. The expression of ACT gene in different organs and during different growth and development stages was basically constant, and when the sequence acted as internal control gene, the semiquantative PCR and RT-PCR have good amplification effect and reproducibility. Conclusion The ACT sequence in E. senticosus is firstly separated and reported, it could act as an internal control gene, and its reaction system of RT-PCR is established.
出处
《中草药》
CAS
CSCD
北大核心
2013年第13期1819-1822,共4页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(30701086)
河北省自然科学基金资助项目(C2009001252)
河北省自然科学基金-石药集团医药联合研究基金项目(H2012401006)
关键词
刺五加
肌动蛋白基因
实时定量PCR
表达稳定性
CDNA克隆
Eleutherococcus senticosus （Rupr. et Maxim.） Maxim.
actin gene
real-time PCR
expression stability
cDNA cloning
