Optimal Reference Genes For Fluorescence Quantitative In Polyploid Of Cyprinus Carpio And Carassius Auratus And Analysis Of Their Expression Stability

Reference genes are usually stably expressed in various cells and tissues.However,it was reported that the expression of some reference genes may be distinct in different species.In this study,based on the molecular biological analysis of transcriptomics technology,RT-PCR and real-time PCR examined their expression levels and systematically evaluated by three software platforms,to answer whether the expression of reported traditional reference genes changes or not in the polyploid fish and optimal reference genes for gene expression analysis in polyploid fish.The main results are as follows:1.12 commonly used reference genes（RPS5,RPS18,RPL7,RPLP2,RPL13α,EF1-α,DDX5,β-actin,β-tubulin,hprt1,B2M,GAPDH）were identified as candidate reference genes by literature review and obtained the m RNA sequencing（RNAseq）data of polyploid fish liver tissue（diploid C.auratus,triploid hybrid and tetraploid hybrid）and obtained the RNAseq data of polyploid fish cultured cells（diploid C.auratus,tetraploid hybrid,and SP4N cells）.By analyzing the transcriptome data of polyploid fish liver tissue,the results showed that RPL7,RPL13α,GAPDH were differentially expressed between tetraploid hybrid and diploid C.auratus（/log₂FC/>1.5 and p-value<0.05）;RPS5,EF1-αbetween tetraploid hybrid and diploid C.auratus p-value<0.05;RPLP2between tetraploid hybrid and diploid C.auratus/log₂FC/>1.5;β-actin between triploid hybrid and diploid C.auratus/log₂FC/>1.5;B2M between triploid hybrid and diploid C.auratus p-value<0.05.By analyzing the transcriptome data of polyploid fish cells,the results showed that RPS18,RPLP2,RPL13α,B2M were between the caudal fin cells of tetraploid hybrid and diploid C.auratus p-value<0.05.Therefore,transcriptome data showed that some traditional reference genes were unstable in polyploid fish.2.The transcriptome data of polyploid fish liver tissue and the transcriptome data of polyploid fish cells indicated that some traditional reference genes were unstable in polyploid fish.Further,10 tissues of three different ploidy fish（diploid C.auratus,triploid hybrid and tetraploid hybrid）and 4 different ploidy fish cell lines（diploid C.auratus caudal fin cells,triploid hybrid caudal fin cells,tetraploid hybrid caudal fin cells,SP4N cell line（C.auratus caudal fin cells induced by the c-Jun N-terminal kinase inhibitor SP600125））were used as materials for RT-PCR.The results showed that in polyploid tissues,among the 12candidate reference genes,except for RPS5,RPS18,andβ-actin,which were expressed to varying degrees in different samples,the other candidate genes all have one or more tissues without obvious positive bands.In polyploid cells,RPS18 andβ-actin were strongly expressed in four polyploid fish cells,the expression of RPS5,RPL13α,RPL7,EF1-α,DDX5,β-tubulin were relatively high in four cell lines,while the expression of RPLP2,hprt1,B2M and GAPDH were low in one or two cells.RT-PCR results showed that there were indeed instability of commonly used reference genes in polyploid fish.3.In order to establish the best reference genes for the study of polyploid fish gene expression,the fluorescent quantitative PCR technology was used to further detected 12 candidate reference genes in10 tissues of three different ploidy fish（diploid C.auratus,triploid hybrid and tetraploid hybrid）and four different ploidy fish cell lines（diploid C.auratus caudal fin cells,triploid hybrid caudal fin cells,tetraploid hybrid caudal fin cells,SP4N cell line）and evaluated the stability of candidate reference genes.In polyploid fish tissues,the Ct values of RPS5,RPS18,and RPL7 have a relatively small variation range,the Ct values of B2M and GAPDH have a large variation range,which preliminarily indicated that the expression of RPS5,RPS18,and RPL7were relatively stable in polyploid fish tissues.In polyploid cells,the Ct values of RPS5,RPS18,and EF1-αvary in a small range,while the Ct values of B2M and GAPDH vary in a large range,which indicated that the expression of RPS5,RPS18 and EF1-αwere relatively stable in polyploid fish cells.4.Based on the Ct value obtained by real-time PCR,the stability of candidate reference genes were systematically evaluated by Best Keeper,Norm Finder and ge Norm three reference gene stability analysis softwares.In polyploid tissues,Best Keeper showed the best stability of RPS18,Norm Finder showed the best stability of RPLP2,and ge Norm showed the best stability of RPS5 and RPS18;In polyploid cells,Best Keeper showed the best stability of EF1-α,Norm Finder showed the best stability of RPS5and RPS18,and ge Norm showed the best stability of RPS5 and RPS18.Due to the differences in the results obtained by different software,a comprehensive ranking of the three softwares showed that in polyploid tissues and polyploid cells,RPS5 ranks first,followed by RPS18.In this study,we confirmed that RPS5 and RPS18 were the most stable reference genes across different tissues and cultured cells for polyploid of fish.These reference genes identified in this study will become useful tools for the molecular biology of polyploid fish.