基于转录组测序的铝胁迫下甘蓝型油菜新内参基因的发掘与引物开发

RNA-seq Based Discovery of New Reference Genes and Primers in Brassica napus under Aluminum Stress

为了研究甘蓝型油菜在铝胁迫条件下内参基因的稳定性,在受到主要铝毒害的根组织内发掘新内参基因,以甘蓝型油菜耐铝品种赣油杂7号和铝敏感品种蓉油18的苗期根组织为研究材料,对营养液培养的试验材料分别设置对照组(0 h)和2个时间梯度(3,24 h)的铝胁迫(100μmol/L AlCl_(3),pH值4.5)处理组。首先,采用有参考基因组的转录组测序技术(RNA-seq)及FPKM值差异基因表达分析,筛选出符合持家基因特征的5个候选内参基因(Actin7、ARFA1E、SAP5、UXS3、UBC9)。随后,依次进行实时荧光相对定量PCR分析,扩增曲线和熔解曲线分析,LinRegPCR扩增效率分析,geNorm、NormFinder和BestKeeper基因表达稳定性分析,REST 2009相对表达量分析等分析步骤。结果表明,新设计开发的5对候选内参基因引物特异性高。从转录组测序数据中筛选出的4个候选内参基因(Actin7、ARFA1E、SAP5、UXS3)的表达稳定性获得了验证。按表达稳定性的综合排名由高至低排序依次为UXS3、SAP5、ARFA1E、Actin7,其中最优组合为SAP5和UXS3。综上所述,本研究开发的候选内参基因引物特异性高,可以提高铝胁迫试验体系实时荧光相对定量PCR试验的准确性,新发掘的内参基因(Actin7、ARFA1E、SAP5、UXS3),可以作为铝胁迫试验体系实时荧光相对定量PCR试验的新内参基因。

In order to study the stability of the endogenous reference genes in Brassica napus under aluminum stress,and discover new reference genes in the root tissues that were mainly poisoned by aluminum,the root tissues of aluminum-tolerant variety Ganyouza 7 and aluminum-sensitive variety Rongyou 18 in Brassica napus at seedling stage were used as the research materials,and the control groups(0 h)and 2 time gradients(3,24 h)of aluminum stress(100μmol/L AlCl_(3),pH 4.5)treatment groups were set respectively in nutrient solution culture.Firstly,five candidate endogenous reference genes(Actin7,ARFA1E,SAP5,UXS3,UBC9)which met the characteristics of housekeeping genes were selected by transcriptome sequencing with reference genome(RNA-seq)and differential gene expression analysis(FPKM).Subsequently,real-time fluorescence relative quantitative PCR analysis,amplification plot and melt curve analysis,amplification efficiency analysis(LinRegPCR),gene expression stability analysis(geNorm,NormFinder,BestKeeper)and relative expression level analysis(REST 2009)were used successively.Results indicated that new primers of candidate endogenous reference genes developed in this study had high specificity.The expression stability of the four candidate endogenous reference genes(Actin7,ARFA1E,SAP5,UXS3)selected from the transcriptome sequencing data had been verified.According to the comprehensive ranking of expression stability from high to low,the order was UXS3,SAP5,ARFA1E,Actin7,and the optimal combination was SAP5 and UXS3.In summary,the new primers of candidate endogenous reference genes developed had high specificity,and could improve the accuracy of real-time fluorescence relative quantitative PCR experiments in aluminum stress experimental system,and newly discovered endogenous reference genes(Actin7,ARFA1E,SAP5,UXS3)could be used in real-time fluorescence relative quantitative PCR experiments of aluminum stress experimental system as new endogenous reference genes.