Bioinformatic Analysis Of Ribosomal Protein Genes In Vertebrates And Their Expression In Teleosts

Ribosomes are molecular machines that perform protein biosynthesis,and ribosome proteins?RPs?are key components of ribosomes.RPs are not only involved in protein synthesis,but also related to other cell processes and functions outside the ribosome,such as regulating gene transcription,mRNA translation,cell proliferation,differentiation and apoptosis independently of the ribosome,playing an important role in the growth and development of organisms.RP genes are the coding genes of RP.In recent years,great progress has been made in the study of the structure and function of RP genes.With more and more RP genes identified from different species,RP genes have become important molecular markers in comparative genomics,genome evolution and phylogeny.However,due to the large number of RP genes,most species have not yet been sequenced,so it is very difficult to understand the complete sequence information and genome structure in different species,and the system evolution is not clear.As the largest group of vertebrates,fish has not been fully identified because of the lack of complete genome sequence in the past.The exact number of RP genes in fish is still unclear.Most of the studies on the expression and function of RP genes in vertebrates focus on a few model animals,and are limited to the study of a single gene.How about the exact number and genomic distribution of RP genes in different taxonomic fish?How about the expression patterns of RP genes in different tissues of teleosts?Do their expression patterns change under the influence of different environmental conditions?These problems have not been solved so far.In this study,the RP genes of 13 representative animals,including Oreochromis niloticus,were identified.At the same time,the expression patterns of RP genes in 8different tissues of Nile tilapia,10 different tissues of southern catfish,4 different stages of gonads in Nile tilapia,5 different stages of gonads in Blue tilapia,3 different stages of gonads in southern catfish,adult gonads of Largescale shoveljaw fish and the gonads treated with different hormones and translation elongation factor genes mutants in Nile tilapia were analyzed by transcriptome analysis.The results are as follows:1)Identification and genome location of RP genes in vertebratesIn this work,we identified 79,64,86,84,83,80,85,125,90,148,88,92 and 86 RP genes in the sea squirt,lamprey,coelacanth,human,mouse,spotted gar,zebrafish,common carp,channel catfish,Atlantic salmon,medaka,tilapia and fugu genome,respectively.These results revealed that the number of the RP genes does not change much in chordates following 2R and 3R events,and significant expansion of RP genes are only observed in teleost fishes with 4R.Amino acid sequence analysis showed that the RP genes of Nile tilapia was 97%,96.5%,94.1%and 92.7%similar to medaka,fugu,zebrafish and human,respectively.The sequences of RP genes were highly conserved among species.It was found that 92 RP genes of Nile tilapia were distributed in 22 linkage groups?LG?,each of which carried one or more genes.Further synteny analysis in other teleosts showed that some RP genes form gene clusters in the genome,which are highly conserved among the Pleuronectiformes,Lampriformes,Perciformes,Gasterosteiformes and Cyprinodontiformes,and also among the Cypriniformes,Siluriformes and Characiformes.The sequence differences of these RP genes on chromosomes can reflect the genetic relationship between Percomorpha and Ostariophysi,which provides a new way to study molecular evolution.2)Phylogenetic analysis of RP genes in vertebratesPhylogenetic and synteny analysis showed that most of the duplicated genes were lost after 2R event,RPL3,RPL7,RPL22 and RPS27 were preserved after 2R,RPS17,RPS27,RPL5,RPL19,RPL22,RPL41 and RPLP2 were preserved after 3R,RPS27 and RPL22 were preserved after 2R as well as 3R.Some genes,such as RPS27,RPL5,RPL7,RPL22 and RPL2,were preserved even after 4R in carp and Atlantic salmon.For the first time,10 duplicated RP genes were found in Nile tilapia.Among them,RPS17,RPS19,RPL3,RPL5,RPL7,RPL19,RPL41 and RPLP2 have two duplicates,RPL22 has three duplicates,RPS27 has four duplicates.In addition,we found a new copy gene of RPS19in Nile tilapia.Duplication of RPS19 was observed only in Percomorpha suggesting that RPS19 may originate from the lineage specific duplication of Percomorpha and be preserved.3)Expression pattern analysis of RP genes in adult teleosts tissuesBased on the transcriptome analysis of 8 different tissues in adult Nile tilapia and 10different tissues in adult southern catfish,we found that all RP genes were expressed in all tissues,but their expression levels were different in different tissues,and their expression levels in the same tissues of different fishes were also different.The highest and lowest expression level was found in the liver and ovary of Nile tilapia,respectively.However,the highest and lowest expression level was found in the ovary and spleen of southern catfish,respectively.In Nile tilapia,the expression level of RPL38 is the highest in brain,heart,kidney,liver and head kidney,and RPS12 is the highest in ovary,testis and muscle;on the contrary,RPL3-2 is the lowest in brain,heart,kidney,liver,testis and ovary,and RPS19b is the lowest in muscle and head kidney.In southern catfish,the expression level of RPL39 is the highest in brain,fin,heart,kidney,head kidney and spleen,RPLP1 is the highest in liver,pancreas and testis,and RPL21 is the highest in ovary;on the contrary,RPL3-2 is the lowest in all tissues.The results of qPCR were consistent with those of transcriptome analysis.Our results further confirmed that RP genes are widely involved in the physiological and biochemical processes of various tissues and cells,and their expression levels can vary with the changes of cell demand and protein biosynthesis demand,reflecting the different metabolic activities of tissues.4)Expression pattern analysis of RP genes in different developmental stages of teleosts gonadsGonads transcriptome analysis of four kinds of teleosts,including Nile tilapia,Blue tilapia,southern catfish and Largescale shoveljaw fish,indicated that the expression level of most RP genes in gonads was high,but there were differences in different sex and different developmental stages,and no sex or development stage specific RP genes were found.The expression patterns of RP genes in the gonads of day after hatch?dah?of Nile tilapia are similar to those in the gonads of 5 and 10 dah of Blue tilapia.The expression levels of RP genes in females are higher than those in males,while in other development stages,the expression patterns of RP genes in males are higher than those in females.In southern catfish,the expression level at 120,360 and 540 dah was higher in males than in females.Further analysis showed that the expression pattern of RP genes was basically the same as that of sex steroid synthetase related genes,and its expression level was positively correlated with the level of sex steroid hormone synthesis.In addition,we found that 11 RP genes in Nile tilapia showed phenotypic sex dependent expression patterns.qPCR and immunohistochemistry analysis showed that the expression of RPL5b in ovary was higher than that in testis,while the expression of RPL24 in testis was higher than that in ovary,which was consistent with the result of transcriptome analysis.The results show that the expression pattern of RP genes in gonad is not invariable,but regulated by sex steroid hormone,which adapts to the variety and quantity of sex steroid hormone synthesis during gonadal development.5)Expression pattern analysis of RP genes in gonads with inhibitors and translation elongation factor mutations in Nile tilapiaGonadal transcriptome analysis of XX fish treated with Fadrozole and RU486 in Nile tilapia,respectively.Compared with normal XX,most RP genes were up-regulated in the gonads of fadrozole_XX and RU486_XX,of which 6?7%?and 64?70%?genes were significantly up-regulated,two?2%?genes was significantly down-regulated,respectively.Compared with normal XY,90?98%?genes were down-regulated in Fadrozole_XX gonad,of which 57?62%?genes were significantly down-regulated and 1gene was significantly up-regulated;53?58%?genes were up-regulated in RU486_XX gonad,only one gene?RPS19b?was significantly up-regulated and one gene?RPL38?was significantly up-regulated.The results showed that the expression pattern of RP genes in gonads treated with different inhibitors was different,and the expression level was affected by different inhibitors.Transcriptome analysis of eEF1A1b^+/-in the testis and 42Sp50^-/-in the ovary indicated that the expression of most RP genes was up-regulated,among which 11 genes were up-regulated in eEF1A1b^+/-testis,29 genes were up-regulated in 42Sp50^-/-ovary,and no down-regulated genes were found.Among them,RPS3,RPL18A and RPL37 were significantly up-regulated in eEF1A1b^+/-testis and 42Sp50^-/-ovary.The results showed that the expression pattern of RP genes in gonads with different mutations of translation elongation factor was different,and the expression level was affected by different mutations of translation elongation factor.The RP genes could compensate for the mutation pressure by up regulating the expression level to maintain the integrity of ribosomal structure and the sustainability and stability of protein synthesis.In conclusion,the number of RP genes in vertebrates does not change much after 2R and 3R,but expands significantly after 4R in teleosts.The distribution of RP genes in the genome of some teleosts is highly conserved,and the differences in the sequence of RP genes can reflect the genetic relationship of fishes with different taxonomic orders.For the first time,92 RP genes were identified in Nile tilapia,and RPS19 was found to be a lineage specific replication gene of Percomorpha.RP genes are widely involved in the physiological and biochemical processes of various tissues and cells,and their expression levels reflect the different metabolic activities of tissues.The expression pattern of RP genes in gonad is basically the same as that of sex steroid synthetase related gene,and its expression level is positively correlated with sex steroid hormone synthesis level.The expression patterns of RP genes in gonads treated with different inhibitors and mutated with different translation elongation factors were different.The expression level of RP genes was affected by different inhibitors and mutations of different translation elongation factors.These results showed that the expression pattern of translation machine of protein biosynthesis—RP genes in teleosts is not constant,but vary with the demand of cell and the demand of the type and quantity of protein biosynthesis.This study enriches the phylogeny and expression cognition of RP genes in vertebrates,especially provides a new perspective for the functional study of protein biosynthesis in the normal and abnormal growth and development of teleosts.