梭梭qRT-PCR内参基因的筛选及稳定性分析  

Screening and Stability Analysis of Internal Reference Genes for qRT-PCR in Haloxylon ammodendron

作　　者：张玲玲[1] 王淑冉 张胜[1,2] ZHANG Lingling;WANG Shuran;ZHANG Sheng(College of Forestry,Northwest A&F University,Yangling,Shaanxi 712100,China;College of Life Science and Technology,Xinjiang University,Urumqi 830046,China)

机构地区：[1]西北农林科技大学林学院,陕西杨凌712100 [2]新疆大学生命科学与技术学院,乌鲁木齐830046

出　　处：《西北植物学报》2023年第12期2005-2017,共13页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金项目(31960333);中央高校基本科研业务费项目(2452023021);新疆维吾尔自治区自然科学基金项目(2019D01C017)。

摘　　要：研究旨在探明梭梭(Haloxylon ammodendron)不同非生物胁迫下稳定表达的内参基因,为后续梭梭抗逆性相关基因功能研究奠定基础。采用实时定量聚合酶链式反应(qRT-PCR)技术从梭梭转录组数据库中检测了GAPDH、EF1-α、UBC、RPL32、ALB、50S-1721、50S-1063、RPⅡ、H3、PP2A、SOD、HSC70、TUA和TUB 14个候选内参基因在热胁迫、干旱、盐、ABA和昼夜节律条件下的表达变化,利用geNorm、NormFinder、BestKeeper和RefFinder软件对梭梭候选内参基因的稳定性进行评价,最终筛选出合适的内参基因,并通过对梭梭磷酸烯醇丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC)基因表达分析,验证不同内参基因对试验结果的影响。4种软件分析得到的最优内参基因存在差异,在RefFinder网站上综合排序分析表明,在ABA处理和昼夜节律下,ALB是最优内参基因,RPⅡ基因在干旱胁迫下表达最稳定,TUB和RPⅡ基因在盐胁迫下最适用,H3基因在热胁迫下表达最为稳定。各种胁迫下最适宜的内参基因为ALB和RPL32。计算几何平均值得14个候选内参基因的综合稳定性排名,其中排名前2位的基因分别为SOD和RPL32。综上,SOD和RPL32可作为梭梭qRT-PCR标准化的内参基因。The aim of this study is to find out the reference genes stably expressed in various abiotic stresses of Haloxylon ammodendron,so as to lay a foundation for the subsequent study on the function of genes related to stress resistance of H.ammodendron.In this study,real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression changes of 14 candidate internal reference genes,including GAPDH,EF-1α,UBC,RPL32,ALB,50S-1721,50S-1063,RPⅡ,H3,PP2A,SOD,HSC70,TUA,and TUB,from the transcriptome database of H.ammodendron,under heat,drought,salt,abscisic acid(ABA)and circadian rhythms.By using geNorm,NormFinder,BestKeeper and RefFinder software,the stability of candidate internal reference genes of H.ammodendron was evaluated,and suitable internal reference genes were finally selected.Furthermore,the expression of phosphoenolpyruvate carboxylase(PEPC)gene was analyzed to verify the influence of different reference genes on the experimental results.Significant differences were found in the optimal internal reference genes ana lyzed by the four software programs,and the comprehensive ranking analysis on the RefFinder website showed that ALB was the optimal internal reference gene under ABA treatment and circadian rhythms,the RPⅡgene was most stably expressed in leaves under drought stress,TUB and RPⅡwere most suit-able internal reference genes under salt stress,and the H3 gene was most stably expressed under heat stress.The most suitable internal reference genes under all stresses were ALB and RPL32.By calculating the geometric mean,the comprehensive stability ranking of 14 candidate internal reference genes was ob-tained,of which the top two genes were SOD and RPL32,respectively.In conclusion,SOD and RPL32 can be used as internal reference genes for qRT-PCR standardization.

关 键 词：梭梭 非生物胁迫 内参基因 QRT-PCR 

分 类 号：Q946[生物学—植物学] S718.3[农业科学—林学]