Studies On The Functional Genes And Regulatory Pathways Related To Skin Color Of Koi Carp (Cyprinus Carpio L.) Based On RNA-Seq

Fish are the most abundant vertebrate group on the earth,with wide distributions and diverse body colors.Different species,or even different individuals of the same species,would show obvious differences in body color in various growth environments or different stages of development.The body color of fish is an important phenotypic trait,with many biological functions like camouflage,alertness,and courtship.Meantime,the color is also a pivotal indicative character for the breeds of the ornamental fish,the quality of colors and pigment patterns directly determines their ornamental and market values.In Addition,the study on differentiations and variations of fish body has been always an important topic in areas of vertebrate biology,biological evolution,and phylogeny,providing ideal models for the exploration of molecular mechanisms on human-related diseases（melanoma,skin cancer,etc.）.The complicated and volatile body colors of fish are affected by various factors,including genetics,nutrition,physiological hormones,and the environment,among which genetics remains the most critical factor.In previous studies on fish body colors,the focus was mostly on growth environment,pigment additives,artificial breeding,and DNA molecular marker identification,while the reports on the mutual regulations of the non-coding RNA and the coding mRNA in pigment cells are relatively scarce.Recently,with the rapid development of high-throughput sequencing technologies,the breadth and depth of research on differentiations and mutations of fish skin colors have been constantly improved and enhanced,thus in-depth exploration of the regulation mechanisms related to the biological processes of pigment cells has important theoretical instructive values for the effective prediction and control of body colors in artificial breeding.Koi carp,a colorful variant of common carp（Cyprinus carpio）,is one of the popular ornamental fish spreading worldwide due to its varied color,slender body,graceful swimming posture,easy feeding,and long-life span.There are many varieties of koi carp,in which Kohaku,Taisho sanke,Showa sanshoku and Utsuri are the most popularly cultured.However,the genetic background of koi carp is relatively complicated and the information of its origin,variation,and evolutionary relationship is incomplete.Currently,in the selection and breeding processes,a traditional method to select a few fish from large numbers of seedlings is still commonly used,which requires intensive labors and financial resources,and has strict requirements on practitioners’ experiences.Therefore,how to stably and efficiently obtain the koi carp with strong body shape,bright color,and well-proportioned pattern distribution to improve its market value has been an enduring goal pursued by culturists.Researchers have carried out a series of work on the body color differentiations and variations of koi carp with respect to nutrition,environment,molecular marker,and gene function analysis.However,there are a few reports on the regulation mechanism of pigment cells in terms of the interactions between non-coding and coding RNA,thus the work in this area is urgently needed.In this study,we analyzed and compared the expression information of lncRNA,mRNA,and miRNA in three different skin body colors（black,red,and white）for koi carp by means of high-throughput sequencing platform.Furthermore,we selected some databases and employed bioinformatics software to perform GO function and KEGG pathway enrichment,the genes and regulatory pathways closely related to differentiations and pigmentations of pigment cells were screened out.Meanwhile,we further investigated the specific mechanisms among these genes through luciferase report assay,qRT-PCR,westernblot,miRNA-antagomir silencing in vivo,lentivirus infection,cell culture,proliferation,migration,and other technologies.The main results are presented as follows:1.The transcriptome sequencing was implemented on three skin color tissues（black,red,and white）by using poly（A）tail enrichment based on Illumina 2000 platform.A total of 590,415,050 clean reads and 446,614 putative transcripts with 4252 known and 72,907 novel lncRNA were simultaneously obtained.Then,92 lncRNA and 722 mRNA with significant differentially expressions（P<0.05）were found through comparisons between the two groups;Ccr_lnc5622441 was found highly expressed in black and red skin,Ccr_lnc14074601 was up-regulated in white skin,whereas pmela,tyr,and other mRNAs were significantly up-regulated（P<0.05）in black skin.Moreover,70 lncRNA acting on 107 target mRNAs in cis and 79 lncRNA acting on 41,625 target mRNAs in trans were also examined.To further reveal their potential functions,Gene Ontology（GO）terms and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathways were analyzed,and it showed membrane,pigment cell development,cAMP signaling,melanogenesis appear to affect skin pigmentation.Finally,the spatio-temporal expression levels of three lncRNA（Ccr_lnc142711,Ccr_lnc17214525 and Ccr_lnc14830101）and three mRNAs（asip,mitf and tyr）involved in the melanogenesis pathway were studied in terms of potential functions in differentiations and migrations of pigment cells in the early embryonic development.2.Through Illumina Hiseq SE50 patterns,we mined the miRNA information for the three skin colors.Firstly,19,649,441,16,193,916 and 18,981,507 clean reads were obtained after the procedures of quality control,filtration,and assembly.Secondly,330,397 and 335 conservative miRNAs（belonging to 81 families）,and 340,353 and 351 candidate miRNAs were screened out by comparing with the database.Thirdly,the family cluster analysis was performed on 20 conservative miRNAs,in which 5 miRNAs（including let-7,miR-124,etc.）shared expression in both protostomes and deuterostomes,13 families（including miR-181,miR-10,etc.）are merely expressed in vertebrate,and the remaining two families（miR-135 and miR-727）have only been identified in fish.Furthermore,A total of 164 differentially expressed（P<0.05）miRNAs（DEMs）was identified,of which 14 have overlapping DEMs in three tissues,including miR-196a,miR-125b,miR-202,etc.Finally,through the target prediction and functional analysis,color-related miRNAs such as miR-200b,miR-206,and miR-196a highlighted putative target genes that are potentially related to pigmentation,including mitf,mc1r,foxd3,and sox10.3.Based on the results from the miRNA sequencing,7 miRNAs with no expression differences were selected for the analysis of internal reference genes.Then,the expression levels of these miRNAs in 15 tissues including blood,kidney,and heart,etc.were analyzed through qRT-PCR,and their stability was evaluated as well by using geNorm,NormFinder and BestKeeper software.The results showed that let-7a is the most stable single reference gene,let-7a and miR-26b are the most stable combination,while 18s rRNA is the most instable one.Finally,selecting let-7a and 18s rRNA as internal reference controls to validate the expression levels of 6 random miRNAs,the results showed that the variation trend is consistent with the sequencing results when let-7a was used as the internal reference,and that has a certain deviation when 18s rRNA was chosen as the internal reference.4.In this chapter,through bioinformatics,RT-PCR,and luciferase reporter assay in koi carp,we concluded that miR-206,a skin-enriched miRNA,regulates mc1r（a key regulator of melanogenesis）expression by binding to its 3’-untranslated region.Then,we silenced miR-206 in vivo with an antagomir method and found a substantial increase of mc1r mRNA expression and its protein level,and its downstream genes:tyr and dct.Moreover,we constructed the miRNA-206 sponge lentivirus vector to transfect koi carp melanocytes in vitro,and further studied the functions of melanocytes using Cck-8 and Transwell assays.As a result,inhibition of miR-206 significantly up-regulated（P<0.05）mc1r mRNA expression and protein level and accelerated the melanocyte proliferation and migration ability compared to the scrambled-sequence negative control groups.5.Based on the results from lncRNA sequencing,4 lncRNA with significantly different expressions were analyzed through qRT-PCR and target gene prediction software,it was found that miR-206 and lnc₁72145 have a complementary relationship.Then,by analysis of the coding ability of lnc₁72145 by CPC,CPAT,and CNIT software,we confirmed that this sequence is lncRNA with no protein coding ability.The luciferase report further demonstrated there is a base complementation between lnc₁72145 and miR-206,indicating that this sequence could be also involved in the regulation of melanogenesis pathway.Finally,through analysis of the spatio-temporal expression level of lnc₁72145,the expression level in eyes and black skins was found to be significantly（P<0.05）higher than that in other tissues,and the gastrulation period became increasing significant（P<0.05）,and this high level remained until 20 days after hatching.