Screening and stable expression analysis of internal reference genes in different tissues of Tulipa gesneriana L.

【Objective】The purpose of the study was to screen the internal reference genes stably expressed in Tulipa gesneriana L. under salt stress conditions,and to provide theoretical references for carrying out the studies related to the regulation of the expression of salt tolerance related genes in T. gesneriana.【Method】T. gesneriana cultivar Red Impression was used as the materials,and it was treated with salt stress at the seedling stage. Roots,bulbs and leaves were collected at different times(0,2,4,6,8,10,12,24 and 48 h),and the expression of candidate internal reference genes in different tissues of T. gesneriana was detected by real-time fluorescence quantitative PCR. The average C_t value for each sample was calculated by Delta CT. Combined with the analysis results of three commonly used software for evaluating the stability of internal reference genes(geNorm,NormFinder and BestKeeper),RelFinder online software was used to comprehensively evaluate the expression stability of candidate internal reference genes and to verify the reliability of the internal reference genes through the expression analysis of the target gene plasma membrane Na⁺/H⁺ reverse transporter protein gene(SOS1)and vesicular membrane Na⁺/H⁺ reverse transporter protein gene(NHX1).【Result】Among the 15 candidate internal reference genes,9 internal reference genes that could be used for real-time fluorescent quantitative PCR in various tissues of T. gesneriana were screened. The results of C_t value analysis of the candidate internal reference genes showed that the average C_t value of 25S rRNA in different tissues was the lowest,the expression abundance was the highest,and the distribution range of C_t value was relatively concentrated,which was an internal reference gene that could be stably expressed in root,bulb and leaf. The results of geNorm and NormFinder analysis showed that 18S rRNA was stably expressed in roots,bulbs and leaves. The 25S rRNA showed good gene stability in roots and leaves,and NADP gene had good gene stability in bulbs. The results of BestKeeper analysis showed that the most suitable internal reference gene in root,bulb and leaf tissues was 25S rRNA,and 18S rRNA had good stability in root and leaf. Combined with the results of the above four analysis,the comprehensive ranking of the expression stability of each candidate gene in different tissues of T. gesneriana was obtained through RefFinder software. Among them,root tissue:25S rRNA>18S rRNA>TUB-6>EF1α>NADP>EF-1α>β-actin;bulb tissue:NADP>25S rRNA>18S rRNA>EF-1α>EF1α>TUB-6;leaf tissue:25S rRNA>18S rRNA>EF-1α>CYP>UBC.【Conclusion】There are differences in the expression of 9 candidate internal reference genes in different tissues of T. gesneriana. 25S rRNA and 18S rRNA are internal reference genes stably expressed in different salt stress treatments in various tissues of T. gesneriana. The use of two internal reference genes at the same time is more conducive to obtaining accurate quantitative results of gene expression.