甘肃特产药材红芪actin基因片段的克隆及序列分析

Cloning and Sequence Analysis of Actin Gene Fragment of Gansu Specialty Medicinal Material Hedysari Radix

目的:研究红芪Hedysari Radix次生代谢产物与基因表达的相关性,克隆红芪的肌动蛋白基因(actin)并进行序列分析和定量研究。方法:通过查找GenBank中的其他豆科植物的编码序列(CDS)设计简并引物,采用逆转录-聚合酶链式反应(RT-PCR)技术克隆红芪actin基因的核心片段,采用实时荧光定量PCR(qRT-PCR)分析该基因在不同部位中的稳定性表达。结果:克隆出了长度946 bp的红芪actin基因,其中编码蛋白框855 bp,编码284个氨基酸,经Blast比对序列,红芪的actin基因与甘草Glycyrrhiza uralensis Fisch.、花苜蓿Medicago ruthenica(L.)Trautv.、红车轴草Trifolium pratense L.的核苷酸序列同源性分别为95.44%、94.42%、94.20%,氨基酸序列的相似性也高达97%以上,红芪actin基因在根、茎、叶不同部位中的表达量基本稳定。结论:首次在国内成功克隆出了红芪的actin基因,并且证实了该基因可作为红芪其他优良基因表达调控的内参基因,为红芪质量研究提供参考。

Objective:To study the relationship between the secondary metabolites and gene expression of Hedysari Radix,actin gene of Hedysari Radix was cloned and carried out sequence analysis and quantitative study.Methods:By searching for CDS sequences of other legume plants in GenBank,we designed degenerate primers,cloned the core fragment of Hedysari Radix actin gene by RT-PCR technology,and analyzed the stable expression of this gene in different organs by real-time fluorescence quantitative PCR(qRT-PCR).Results:The actin gene of Hedysari Radix with a length of 946 bp was cloned,of which 855 bp encodes a protein box and encodes 284 amino acids.After blast alignment,the Actin gene of Hedysari Radix has 95.44%,94.42%and 94.20%homology with the nucleotide sequences of Glycyrrhiza uralensis Fisch.,Medicago rutenica(L.)Trautv.and Trifolium pratense L.respectively.The similarity of amino acid sequence is over 97%,the expression level of Hedysari Radix actin gene in different organs was basically stable.Conclusion:This study successfully cloned the actin gene of Hedysari Radix for the first time in China,and it was confirmed that this gene can be used as an internal reference gene for regulating the expression of other excellent genes of Hedysari Radix,laying a foundation for the research of quality and efficiency improvement of Hedysari Radix.