Using the transcriptome to evaluate the best reference genes for studying nutrition of the critically endangered Yangtze sturgeon (Acipenser dabryanus)

Abstract

Abstract To better apply qRT-PCR in nutrition research, this study screened the reference genes (RGs) of the Yangtze sturgeon (Acipenser dabryanus) using the transcriptome. The results revealed that transcriptome sequencing of the liver, anterior gut (esophagus, cardia stomach, pyloric stomach), and posterior gut (pyloric caecum, duodenum, valvular gut, rectum) identified 179,199 unigenes with an N50 of 1747\u202fbp and average length of 1127\u202fbp. In the transcriptome, 19 unigenes with coefficients of variation 400 were screened. Excluding unannotated and repetitive unigenes, 9 potential reference genes were obtained, namely mitochondrial phosphoenolpyruvate carboxykinase (mPEPCK), Tc1 transporase (Tc1Tase), β-actin, elongation factor 2 kinase (EF2Kase), myeloid leukemia cell differentiation protein (Mcl1), histone H3.3 (hH3.3), elongation factor 1-alpha (EF1-α), thioredoxin interacting protein (txnip) and cofilin-2-like (CF2). In addition, 6 empirically commonly used RGs were also identified as potential candidates, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUBB), β 2-microglobulin (B2M), succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), hypoxanthine phosphoribosyltransferase 1 (HPRT1), and 18S rRNA. We designed the qRT-PCR primers of potential RGs and plotted a standard curve. The amplification efficiency of all genes is more than 91.5% (91.5–96.7%). Comprehensive analysis using Delta Cq, Bestkeeper, Normfinder and geNorm revealed that the expression stability of potential RGs is EF1-α\u202f>\u202fhH3.3\u202f>\u202fCF2\u202f>\u202fβ-actin >18S rRNA >B2M\u202f>\u202fHPRT1\u202f>\u202fGAPDH >mPEPCK >TUBB >Mcl1\u202f>\u202fSDHA >EF2Kase\u202f>\u202ftxnip >Tc1Tase in postprandial, EF1-α\u202f>\u202fβ-actin >hH3.3\u202f>\u202fCF2\u202f>\u202fHPRT1\u202f>\u202fB2M\u202f>\u202fmPEPCK >18S rRNA >SDHA >TUBB >GAPDH >Mcl1\u202f>\u202fEF2Kase\u202f>\u202ftxnip >Tc1Tase in fasting and refeeding, and β-actin >GAPDH >EF1-α\u202f>\u202fCF2\u202f>\u202fhH3.3\u202f>\u202fB2M >18S rRNA >HPRT1\u202f>\u202fTUBB >Mcl1\u202f>\u202fmPEPCK >EF2Kase\u202f>\u202fTc1Tase\u202f>\u202fSDHA >Txnip during weaning. The weighted analysis of postprandial, fasting and refeeding, and weaning indicated that the stability of potential RGs is EF1-α\u202f>\u202fβ-actin >hH3.3\u202f>\u202fCF2\u202f>\u202fB2M >18S rRNA >HPRT1\u202f>\u202fGAPDH >mPEPCK >TUBB >Mcl1\u202f>\u202fSDHA >EF2Kase\u202f>\u202ftxnip >Tc1Tase. In summary, EF1-α and/or β-actin are recommended as RGs when qRT-PCR is used in the nutrition research of Yangtze sturgeon.