沙芥qRT-PCR内参基因的筛选与验证  

Screening and validation of reference genes in qRT-PCR of Pugionium cornutum

作　　者：武兆昕 商凯凡 王萍[1] WU Zhaoxin;SHANG Kaifan;WANG Ping(College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Hohhot 010018,China;Institute of Vegetables and Flowers,Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China)

机构地区：[1]内蒙古农业大学园艺与植物保护学院,呼和浩特010018 [2]内蒙古自治区农牧业科学院蔬菜花卉研究所,呼和浩特010031

出　　处：《植物生理学报》2025年第3期359-370,共12页Plant Physiology Journal

基　　金：国家自然科学基金(31860553和31260475)。

摘　　要：从沙芥全长转录组数据中选择10个内参基因(TIP41、UBQ10、UBC9、UP1、SAND、GAPDH、ACT、EF1α、PP2A、TUB)作为候选内参基因,使用qRT-PCR法检测候选内参基因在沙芥非生物和植物生长调节物质处理下的表达水平,并使用GeNorm、Normfinder、BestKeeper和RefFinder分析其稳定性。结果表明:EF1α为沙芥所有处理样本混合组(Total组)最稳定内参基因;TIP41为干旱叶/根、NaCl处理叶根组合(LR组)/根、脱水LR组/叶片、ABA处理LR组/叶片、MeJA处理LR组/根中最稳定的内参基因;UBQ10为4℃处理LR组/叶片、42℃处理LR组、干旱LR组及SA处理LR组最稳定内参基因;ACT为4℃处理根、脱水处理根中最稳定内参基因;GADPH为42℃处理叶片中最稳定内参基因;PP2A为NaCl处理叶片、ABA处理根、SA处理根中最稳定的内参基因;UBC9为MeJA处理叶片中最稳定的内参基因。此外,选择3个胁迫相关基因(PcDREB2、PcCLCg和PcHSP17.8)验证了内参基因的准确性。本研究结果可用于沙芥非生物胁迫和植物生长调节物质处理下qPCR分析,为今后沙芥耐逆基因的表达分析提供了参考基因和理论依据。In this study,10 reference genes(TIP41,UBQ10,UBC9,UP1,SAND,GAPDH,ACT,EF1α,PP2A,and TUB)were selected as candidate reference genes from the full-length transcriptome data of Pugionium cornutum.The expression levels of 10 genes in abiotic and hormone treatments were detected by qRTPCR,and their stability was analyzed by GeNorm,Normfinder,BestKeeper and RefFinder.EF1αwas the most stable reference gene in the mixed group of all treated samples(Total).TIP41 was the most stable reference gene in drought leaves/roots,leaf and root combination(LR group)/roots under NaCl stress,LR group/leaves under dehydration treatment,LR group/leaves under ABA treatment,and LR group/roots under MeJA treatment.UBQ10 was the most stable reference gene in LR group/leaves under 4℃stress,LR group under heat stress,LR group under drought and SA treatment.ACT was the most stable reference gene in roots under 4℃and dehydration stress.GADPH was the most stable reference gene in leaves under heat stress.PP2A was the most stable reference gene in leaves under NaCl stress,roots under ABA and SA treatment.UBC9 was the most stable reference gene in leaves under MeJA treatment.Furthermore,the accuracy of selected reference genes was verified by normalizing the stress-related genes(PcDREB2,PcCLCg and PcHSP17.8)expression.These suitable reference genes may be used in future qRT-PCR analyses of P.cornutum tissues under different abiotic stresses and hormones,which provided a reference gene and theoretical basis for the expression analysis of stress tolerance genes in P.cornutum for the future study.

关 键 词：内参基因 沙芥 非生物胁迫 植物生长调节物质 QRT-PCR 

分 类 号：Q943.2[生物学—植物学]