Comprehensive Transcriptome Analysis Reveals Four Reproductive Development Related Genes In Spinyhead Croaker(Collichthys Lucidus)

Spinyhead croaker（Collichthys lucidus,C.lucidus）,an important small demersal economic fish,is widely distributed in estuaries and coastal areas of China.With its tender and delicious meat,and can be captured all year round,spinyhead croaker is popular with coastal residents.However,the limited population and reproduction rate of spinyhead croaker are difficult to satisfy the demand of market,and there’s a risk of overfishing and population decline.We have carried out a systematic study on the genetic structure of the population of C.lucidus,and are gradually carrying out a research on large-scale breeding technology of C.lucidus.At present,the transcriptome information of C.lucidus remains largely unknown,,and the cytological and reproductive studies of C.lucidus have not been reported.The research of the germ cells development and the related regulatory mechanism on C.lucidus might has profound significance for the in-depth and systematic study of genetic basis and the industrial development of C.lucidus.In this study,reproductive development related genes and their signaling pathway were analyzed by de novo transcriptome assembly of the adult testes and ovarian of C.lucidus,using Illumina HiSeq^TM 500 platform.Moreover,the full-length sequences of germ cell specific genes,including dnd,dmrt1,foxl2 and vasa,were cloned,and their expression patterns and functions were analyzed.Finally,the reproductive development related microRNAs were predicted by database.On the one hand,the results of this study might enrich the transcriptome information of the database and provide a theoretical basis for further study in the mechanism of germ cell development of C.lucidus.On the other hand,it might provide an important basis for the protection and rational development of the germplasm resources in C.lucidus.More details are as follows:1.Transcriptome sequencing and differential gene analysis of C.lucidusUsing the Illumina NextSeq^TMM 500 platform,adult testes and ovarian transcripts of C.lucidus were assembled by de novo,differentially expressed genes associated with sex determination,germ cell development and other processes were identified.The clean reads identified in testis and ovary were 57,087,688 and 60,867,978,respectively,including 189,181 transcripts and 131,168 unigenes.By mapping the transcripts to NR,GO and KEGG Pathway databases,it was found that:the genetic sequences of C.lucidus were the most similarly to Maylandia zebra gene（26%）.A total of 27,708 genes were annotated by GO function and 650 genes were related to the reproduction and reproductive process.There were 12,883 unigenes mapped to the KEGG Pathway,of which the"signal transduction mechanism"pathway enriched the most unigenes,accounting for 14.6%.Twelve gonadal specific genes were selected from transcriptome sequencing data,and their expressions in C.lucidus adult tissues were analyzed by fluorescence quantitative PCR（qPCR）.The results were consistent with the sequencing results.2.Molecular cloning and expression pattern analysis of dnd/dmrt1/foxl2/vasa in C.lucidus gonadsDnd,dmrt1,foxl2 and vasa were selected from the differentially expressed genes in the previous chapter for further analysis.The full-length cDNA sequences of dnd,dmrt1,foxl2 and vasa were obtained by RACEs.The four genes were highly conserved in structure and evolutionary relationship.The results of qPCR showed that dmrt1 mRNA was only detected in the testis,but not in the other somatic tissues,including ovary.Foxl2mRNA was expressed in the brain,heart and ovary,but no not in the testis.Both Dnd and vasa mRNA were specifically expressed in gonads and there was no significant difference in the expression level between testis and ovary.To determine the subcellular localization of four genes in the adult gonads of C.lucidus,in situ hybridization（ISH）was performed.It was found that dmrt1 signal was detected in the secondary spermatocytes,sperms and Leydig cells of the testis,but not in the ovary.Foxl2 was localized in oocytes and granulosa cells of the ovary,while no signal was detected in the testis.The results of ISH showed obvious gender dimorphism,which was consistent with that of qPCR,indicating that dmrt1 and foxl2 might play vital roles in the development of testis and ovary,as well as the process of sex differentiation.Vasa and dnd were both expressed in the cytoplasm of germ cells with similar expression patterns.In the testis,vasa/dnd signals were highest in spermatogonia and decreased gradually with spermatogenesis,and the signal finally disappeared in spermatozoa.In the ovary,vasa/dnd signals showed a trend of increase decrease.The RNA signal of vasa or dnd was weak in the oocyte,and became strong in the early oocyte,then was lost in the late oocyte and eggs.Dual color fluorescence in situ hybridization（FISH）was used to compare the subcellular localization differences of vasa/dnd.Interestingly,the results revealed that vasa singal showed a specific expression with its signal intensified and patched in the central region of stage I oocytes,which was different from dnd.Furthermore,the results suggested that both vasa and dnd genes not only played different important roles in the development of germ cell,but also showed synergies effects.In addition,the recombinant plasmids of vasa and dnd 3’UTR fused with fluorescent protein were constructed and transcribed into mRNA.Localized RNA expression（LRE）showed that 3’UTR of vasa and dnd could induce the stable expression of green fluorescent protein（GFP）in germ cells and successfully visualize the PGCs of medaka.They have the conservative function of marker gene,which can be used to observe the migration of PGCs in embryo.3.Prediction of reputative development related microRNA in C.lucidusThe miRNAs（microRNAs）were predicted by database of miBase,miRWalk and TargetSCAN.Results revealed both 3’UTR of vasa and dnd displayed a site that was complementary to miR-430 conserved seed sequences（CGUGAAA）.The dnd/dmrt1/foxl2/vasa mRNA sequences were used to predict the miRNAs that might be involved in the post-transcriptional regulation of reproductive development.Subsequently,the expression of candidate miRNAs in adult tissues were verified by qPCR,and 6 gonadal specific miRNAs were obtained.Among them,mir-10-38 and mir-54 were only expressed in testis.The expressions of mir-143-5,mir-202-2 and mir-43-4 in the testis were significantly higher in the testis than those in the ovary.Moreover,mir-1260b-1 was mainly expressed in the ovary.Further study should be conducted in the targeted genes and functions of these miRNAs.