土家族药血筒实时荧光定量PCR内参基因的筛选和验证  

Selection and validation of reference genes for quantitative real-time PCR analysis in Tujia medicine Xuetong

作　　者：肖倩 谭琛偲 曾江 徐媛菽[1,2] 符天昊 宁露云 王炜 XIAO Qian;TAN Chen-si;ZENG Jiang;XU Yuan-shu;FU Tian-hao;NING Lu-yun;WANG Wei(School of Pharmacy,Hunan University of Chinese Medicine,Changsha 410208,China;Traditional Chinese Medicine and Ethnomedicine Innovation&Development International Laboratory,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学药学院,湖南长沙410208 [2]湖南中医药大学中医药民族医药国际联合实验室,湖南长沙410208

出　　处：《中国中药杂志》2025年第3期682-692,共11页China Journal of Chinese Materia Medica

基　　金：湖南省自然科学基金项目(2023JJ40482);湖南省教育厅科学研究项目(22C0189);2022年度中医药民族医药国际联合实验室开放基金项目(2022GJSYS09);湖南中医药大学本科生科研创新项目(2023BKS115,2024BKS079)。

摘　　要：土家族药血筒(植物学名:异形南五味子)以木质藤本茎入药,具有抗类风湿性关节炎、保肝、抗肿瘤、抗氧化的药用价值,是湖南、广东等地的常用药材。筛选合适稳定的内参基因是开展异形南五味子分子水平研究的基础。该研究选取GAPDH、TUA、Actin、UBQ、EF-1α、18S-rRNA、CYP、UBC、TUB、H2A、RPL共11个基因作为候选内参基因,通过实时荧光定量PCR检测11个候选内参基因在异形南五味子6个不同组织部位(藤茎-形成层以内、藤茎-形成层以外、果实、花、根和叶)与不同干预条件下[干旱胁迫、盐胁迫、茉莉酸甲酯(MeJA)处理]的基因表达水平,利用geNorm、NormFinder、ΔCT算法以及RefFinder软件对11个候选内参基因表达稳定性进行综合分析评价。综合以上分析结果表明,UBC与RPL基因在6个不同组织部位中较为稳定,UBC与GAPDH基因在不同干预条件下较为稳定。为了验证异形南五味子内参基因的可靠性,该研究进一步检测了异形南五味子主要成分——血筒素合成通路上的14个相关基因(KhFPS、KhIDI、KhCAS、KhSQE、KhSQS、KhSQS-2、KhHMGS、KhHMGR、KhMVD、KhMVK、KhDXR、KhDXS、KhPMVK、KhGGPS)在不同组织与不同干预条件中的表达水平。结果显示,分别以UBC与RPL或UBC与GAPDH作为内参基因,14个基因在异形南五味子不同组织部位或不同干预条件下表达趋势基本一致。综上,该研究筛选的UBC基因可作为异形南五味子不同组织部位与不同干预条件下的内参基因,为后续开展异形南五味子主要成分生物合成途径的研究奠定了基础。Tujia ethnic group medicine Xuetong is derived from Kadsura heteroclita,the stem of which has the medicinal value for anti-rheumatoid arthritis,liver protection,anti-tumor,anti-oxidation effects,and has been widely used in Hunan and Guangdong in China.The selection of reliable and stable reference genes is the basis for subsequent molecular research on K.heteroclita.In this study,GAPDH,TUA,Actin,UBQ,EF-1α,18S-rRNA,CYP,UBC,TUB,H2A,and RPL were selected as candidate reference genes in Kadsura heteroclita.The gene expression levels of the 11 candidate reference genes of K.heteroclita in its 6 different parts(stem-inside of the cambium,stem-outside of the cambium,fruit,flower,root,and leaf)and under different intervention conditions[drought stress,salt stress,and methyl jasmonate(MeJA)treatment]were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The expression stability of the 11 candidate reference genes was comprehensively analyzed and evaluated by geNorm,NormFinder,ΔCT algorithm,and RefFinder software.The results showed that the expression of UBC and RPL was relatively stable in 6 different parts,and UBC and GAPDH genes were relatively stable under different intervention conditions.To verify the reliability of reference genes for K.heteroclita,this study further examined the relative expression levels of KhFPS,KhIDI,KhCAS,KhSQE,KhSQS,KhSQS-2,KhHMGS,KhHMGR,KhMVD,KhMVK,KhDXR,KhDXS,KhPMVK,and KhGGPS in different parts and under different intervention conditions,which might relate to the synthesis of the main component(Xuetongsu)of K.heteroclita.The results showed that with UBC and RPL or UBC and GAPDH as the reference genes,the expression trends of these 14 genes were basically consistent in different parts or under different intervention conditions for K.heteroclita.In conclusion,UBC can be used as a reference gene of K.heteroclita for its different parts and different intervention conditions,which lays a foundation for further research on the biosynthetic pathway of main components in K.het

关 键 词：异形南五味子 实时荧光定量PCR 内参基因 UBC 

分 类 号：S567.19[农业科学—中草药栽培]