摘要
[目的]筛选楸树在非生物胁迫和不同发育阶段的稳定内参基因,提高实时荧光定量PCR基因表达检测结果的准确性。[方法]基于课题组前期基因芯片技术检测的基因表达数据,筛选了10个候选内参基因(TUA、ACTIN、GAPDH、UBI、RPL、RPS16、SUV、UBC、CRC和ABC)。通过qRT-PCR检测了这些基因在楸树幼苗不同萌发阶段的叶和根,以及ABA、NaCl、PEG、冷胁迫处理后叶片、根的表达量,运用genorm、Normfinder、Bestkeeper和ΔCT 4种方法评价表达稳定性。[结果]熔解曲线分析表明,10对引物均呈现单一特异性峰。QRT-PCR检测显示,候选内参基因在不同组织和处理中的平均值CT变幅为20.19~30.51,其中UBI的CT值最低(表达量最高),ABC的CT值最高(表达量最低)。胁迫处理分析表明,叶片中,CRC、UBC和GAPDH在3种以上评价方法中排名前4位,为叶片最适内参基因;根中,GAPDH和UBC在4种方法排序均排前4位,为根最适内参基因。发育阶段分析显示,不同发育阶段叶片和根中,RPS16和GAPDH稳定性在4种方法均排前4位,是楸树幼苗发育研究的最适内参基因。[结论]本研究首次系统地为多基因型混合的楸树幼苗在非生物胁迫及不同发育阶段的qRT-PCR分析提供了全面可靠的内参基因,同时为紫葳科乃至其他阔叶树种的基因表达研究提供参考。
[Objective]To identify stable reference genes in Catalpa bungei under abiotic stress and different developmental stages,thereby improving the accuracy of gene expression analysis using Quantitative Reverse-Transcriptase Polymerase Chain Reaction(qRT-PCR).[Method]Based on data obtained from gene chip technology in our previous study,ten candidate reference genes(α-Tubulin(TUA),ACTin-7(ACTIN),Glyceraldehyde-3-phosphate dehydrogenase(GAPDH),polyubiquitin(UBI),ribosomal protein L2(RPL),30S ribosomal protein S16(RPS16),mitochondrial RNA helicase(SUV),Putative ubiquitin-conjugating enzyme(UBC),Chromatin remodeling Complex subunit(CRC)and ABC transporter family protein(ABC))were selected.Expression levels of these genes were quantified via qRT-PCR in leaves and roots of Catalpa bungei seedlings at different germination stages,as well as in leaves and roots subjected to ABA,NaCl,PEG,and low-temperature stress treatments.Expression stability was evaluated using four independent algorithms:geNorm,NormFinder,BestKeeper,andΔCT.[Results]Melting curve analysis demonstrated that all 10 primer pairs exhibited single specific peaks.QRT-PCR results revealed that the average CT values of candidate reference genes across different tissues and treatments ranged from 20.27 to 30.53,with TUA showing the lowest Ct value(indicating the highest expression level)and ABC displaying the highest Ct value(indicating the lowest expression level).Stress treatment analysis indicated that in leaves,CRC,UBC,and GAPDH ranked within the top four positions across three or more evaluation methods,identifying them as the optimal reference genes for leaf tissues.In roots,GAPDH and UBC consistently ranked in the top four across all four methods,making them the most stable reference genes for root tissues.Across developmental stages,RPS16 and GAPDH exhibited the highest stability(ranking within the top four positions across all four methods)in both leaves and roots at different developmental stages,making them the optimal reference genes for studying Catalpa bungei seedling development.[Conclusion]This study is the first to systematically identify and validate comprehensive and reliable reference genes for qRT-PCR analysis in multi-genotype mixed Catalpa bungei seedlings under various abiotic stress and developmental stages.The findings provide a critical framework for gene expression studies in Bignoniaceae species and other broad-leaved tree taxa.
作者
王茂鹏
刘莹
胡继文
卢楠
张苗苗
王楠
王军辉
高彩球
麻文俊
WANG Mao-peng;LIU Ying;HU Ji-wen;LU Nan;ZHANG Miao-miao;WANG Nan;Wang Jun-hui;Gao Cai-qiu;MA Wen-jun(National Key Laboratory of Forest Tree Genetics and Breeding,College of Forestry,Northeast Forestry University,Harbin 150040,Heilongjiang,China;Research Institute of Forestry,Chinese Academy of Forestry,Beijing 100091,China)
出处
《林业科学研究》
北大核心
2025年第6期107-118,共12页
Forest Research
基金
“十四五”国家重点研发计划(2021YFD2200301)。
