工业大麻种子萌发及渗透胁迫下实时荧光定量PCR内参基因的筛选  

Selection of reference genes for quantitative real-time PCR in seed germination and osmotic stress conditions in industrial hemp

作　　者：张涵雪 张达 胡建明 汤开磊 杜光辉[1] ZHANG Han-xue;ZHANG Da;HU Jian-ming;TANG Kai-lei;DU Guang-hui(Institute of Resource Plants,Yunnan University,Kunming 650500,Yunnan,China)

机构地区：[1]云南大学资源植物研究院,云南昆明650500

出　　处：《云南大学学报（自然科学版）》2023年第3期733-740,共8页Journal of Yunnan University(Natural Sciences Edition)

基　　金：国家自然科学基金(32160514);国家麻类产业技术体系建设专项(CARS-16-E15);云南省教育厅科学研究基金(2022J0015);云南大学研究生科研创新项目(2021Z120).

摘　　要：为了筛选适用于工业大麻种子正常萌发与渗透胁迫下萌发的实时荧光定量PCR(qRT-PCR)内参基因,研究以‘云麻7号’种子为试验材料,设置正常萌发和20%PEG渗透胁迫下萌发处理.利用qRT-PCR技术对10个候选参考基因(CDPK,eIF4E,TIP41,UBQ,EF1α,PP2A,Actin,Clathrin,TUB,DHS2)在3个萌发时间点(萌发3、5、7 d)幼苗中的表达量进行检测.通过GeNorm、Normfinder和BestKeeper三种软件包对内参基因表达稳定性进行评价,再利用RefFinder在线软件进行综合分析,从而获得稳定、适宜的内参基因.结果表明,在工业大麻种子正常萌发过程中,PP2A基因的稳定性最好.在渗透胁迫下的种子萌发中,UBQ基因的稳定性最好.综合两组结果得出,eIF4E内参基因的表达最稳定.研究结果确定了工业大麻种子萌发及渗透胁迫下进行qRT-PCR分析的最适内参基因,为今后研究工业大麻种子萌发响应渗透胁迫相关基因的表达提供前期基础.In order to screen the stable reference genes for quantity real-time PCR suitable in hemp seed germination and osmotic stress conditions,10 candidate reference genes(CDPK,eIF4E,TIP41,UBQ,EF1α,PP2A,Actin,Clathrin,TUB,DHS2)were analysed in this study.‘Yunma 7’seeds were treated with distilled water(normal germination)and 20%PEG(osmotic stress),respectively.Seedling samples were taken at 3 different seed germination periods(3 d,5 d and 7 d after treatment).The expression of candidate reference genes was detected by qRT-PCR technology,and the expression stability was evaluated by GeNorm,Normfinder and BestKeeper software packages.The appropriate reference genes were obtained by comprehensive analysis of RefFinder online software.Among 10 candidate reference genes,the stability of PP2A gene was the best in the normal germination stage of hempseed,the stability of UBQ gene was the best in seed germination under osmotic stress,and the expression of eIF4E(as a reference gene)was the most stable under these two conditions.In this study,the most suitable reference genes for qRT-PCR analysis of hempseed germination under osmotic stress were determined,which laid a foundation for the expression analysis of genes related to seed germination under osmotic stress of hemp in the future.

关 键 词：工业大麻 种子萌发 渗透胁迫 内参基因 实时荧光定量PCR 

分 类 号：S563.3[农业科学—作物学]