剑麻内参基因筛选与稳定表达分析

Screening of Suitable Reference Genes for qRT-PCR Normalization in Sisal

为了筛选剑麻中稳定表达的内参基因,保证基因表达结果的可靠性,本研究以热麻1号为材料,采用实时荧光定量RT-PCR方法对来自剑麻转录组数据的8个内参基因(EF1α、MADH、ACT2、GAPDH、TUB、ACT54、CYP和EF1β)在烟草疫霉侵染、水杨酸(SA)、茉莉酸甲酯(MeJA)和脱落酸(ABA)处理过程中的表达水平进行检测,并结合GeNorm、BestKeeper和NormFinder软件综合评价8个内参基因的稳定性。结果表明:8个内参基因在不同处理下的表达稳定性存在显著差异,其中在烟草疫霉侵染和茉莉酸甲酯处理过程中,EF1α表达最稳定,EF1β表达最不稳定。在水杨酸处理过程中,EF1α表达最稳定,ACT2表达最不稳定。在脱落酸处理过程中,GAPDH表达最稳定,ACT54表达最不稳定。该结果为剑麻基因的表达分析提供了可供选择的内参基因。

In this study,eight reference genes(EF1α,MADH,ACT2,GAPDH,TUB,ACT54,CYP and EF1β)from the transcriptome database,were selected and analyzed in Rema No.1 including Phytophthora nicotianae Breda,MeJA,SA and ABA treatments.Statistical tools,including GeNorm,NormFinder and BestKeeper,were utilized to assess the suitability of reference genes based on the stability rankings for different species.The stability of the eight reference genes was significantly different under different treatments in Rema No.1.For Phytophthora nicotianae Breda and MeJA stresses,EF1αwas identified as the most stable gene and EF1βwas identified as the least stable gene.For SA stress,EF1αwas identified as the most stable gene,and ACT2 was identified as the least stable gene.For ABA stress,GAPDH was identified as the most stable gene,and ACT54 was identified as the least stable gene.The results would provide reliable and optional available reference genes in gene expression analysis of sisal.