Selection Of Reference Genes After Different Conditions Stimulation In Golden Pompano(Trachinotus Ovatus)

The reference genes,also known as the housekeeping genes,are the stable expression genes in all cells.The expression levels of this genes are less influenced by external factors,and are expressed continuously in most tissues of each growing stage.Golden pompano(Trachinotus ovatus)is an important economic fish and the main species for offshore cage aquaculture in Hainan and other southern coastal areas of China.In this study,we aim to confirm the reliability reference genes in Golden pompano after different stimulation through quantitative real-time PCR(qRT-PCR).The PCR data were analyzed using the geNorm and NormFinder algorithms.The results of this study will provide a reference for the immunological studies of T.ovatus.And it lays a theoretical foundation for the follow-up real-time fluorescence quantitative PCR in reference genes.To detect the expression of the reference genes,eight tissues including spleen,head kidney,liver,gill,intestine,brain,muscle and heart,were selected from T.ovatus which were stimulated by poly I:C.The results showed that there were differences in the expression of the selected eight housekeeper genes in different tissues.When under normal physiological condition,geNorm and NormFinder analyses B2M and 18S rRNA could be the appropriate genes.GeNorm results show that,after 12h of stimulation with poly I:C,Actin/B2M,B2M/UBCE,Actin/UBCE,Actin/UBCE,RPL13/B2M,B2M/UBCE,B2M/TTLL1 and UBCE/GAPDH,as the most stably expressed genes in spleen,kidney,liver,gill,intestine,brain,muscle and heart,respectively.After 48h stimulation with poly I:C,B2M/RPL13,B2M/RPL13,UBCE/TTLL1,Actin/EFIA,RPL13/B2M,RPL13/B2M,Actin/GAPDH and TTLL1/RPL 13 were ranked as the most stably expressed genes in spleen,kidney,liver,gill,intestine,brain,muscle and heart,respectively.Reference genes received similar rankings when NormFinder was used to conduct the analysis.In a word,B2M and 18S rRNA are the most stable reference genes under normal physiological conditions.However,after poly Ⅰ:C stimulation,no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types.In selecting a preferred gene,B2M,B2M,UBCE,Actin,B2M/RPL13,B2M,B2M and RPL13 can be used in spleen,kidney,liver,gill,intestine,brain,muscle and heart,respectively.The expression of the eight selected genes in four immune organs stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data were analyzed using the geNorm and NormFinder algorithms.The results showed when studying gene expression in response to bacterial stimulation,the selection of the internal controls should be tissue specific.After 48h of stimulation with V.harveyi,geNorm ranked EF1A/Actin,18S rRNA/B2M,UBCE/B2M,and 18S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48h of stimulation with E.tarda,geNorm ranked 18S rRNA/EF1A,18S rRNA/B2M,B2M/RPL13,and 18S rRNA/EF1A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48h of stimulation with S.agalactiae,18S rRNA/EF1A,18S rRNA/B2M,B2M/Actin,and 18S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively,by geNorm.NormFinder results show that,After 48h of stimulation with V.harveyi,F1 A,18S rRNA/B2M,UBCE/B2M,18S rRNA,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48h of stimulation with E.tarda,UBCE,18S rRNA/B2M,B2M/RPL13 and 18S rRNA,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48h of stimulation with S.agalactiae,18S rRNA/EF1A,B2M,B2M/Actin and 18S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.However,the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.In selecting a preferred gene when infected by bacterial,EF1A,B2M,B2M,18S rRNA can be used in spleen,kidney,intestine and gill,respectively,of T.ovatus.