Study On The Function Of WWOX Gene In Melanin Synthesis Of Lanping Black-Boned Sheep

In recent times,black foods including melanin,anthocyanins,and flavonoids,have garnered considerable attention and have been called one of the“super-nutrients”that are deemed indispensable for the human body and are even incorporated into dietary fiber supplements.Although anthocyanins and flavonoids are commonly present in plant-based foods,melanin is chiefly found in select food items such as black rice,black sesame,black bone chicken,and the singular mammal,Lanping Black-boned Sheep,which has superior nutritional value due to its high melanin content.The present study delves into the underlying molecular mechanism of melanin biosynthesis in Lanping Black-boned Sheep and the functional genes related to melanin were identified to provide a realistic basis for the development,these findings have significant implications for the nutritional utilization,conservation,and development of the distinctive local breed and nutritionally rich Lanping Black-boned Sheep.The WW domain containing oxidoreductase（WWOX）gene,identified as a candidate melanogenic gene through whole-genome resequencing and transcriptome analysis,was investigated in this study.Various approaches were taken to explore the function of the WWOX gene in melanin synthesis of Lanping Black-boned Sheep,including measurement of TYR activity,pheomelanin,eumelanin,alkaline-soluble melanin,total melanin,and plasma colorimetric indexes.Molecular cloning of the CDS region of the gene and bioinformatic prediction of the WWOX protein were also conducted.Additionally,9 exons of the WWOX gene were PCR amplified to obtain polymorphic mutation sites,which were then correlated with 6 melanin content indexes.Finally,the spatiotemporal expression and mutant phenotype of the WWOX gene was investigated using both whole-mount in situ hybridization of zebrafish and CRISPR/Cas9 technique.Moreover,four melanin genes in this mutant were studied to gain further insight into the expression of WWOX in melanin synthesis,and the main experimental results were as follows:1.The TYR activity in plasma of Lanping Black-boned Sheep and ordinary sheep from Lanping and Huize regions was 496.164±25.313 IU/m L、312.741±20.967 IU/m L and 318.594±11.235 IU/m L respectively.The content of pheomelanin is 0.259±0.087 OD/m L,0.172±0.044 OD/m L and 0.204±0.0526 OD/m L respectively.The eumelanin is 0.101±0.015 OD/m L,0.083±0.014 OD/m L and0.082±0.014 OD/m L.The alkaline-soluble melanin is 0.196±0.046 OD/m L,0.103±0.023 OD/m L,0.188±0.032 OD/m L.The plasma colorimetric is 0.139±0.071OD/m L,0.071±0.028 OD/m L,0.067±0.022 OD/m L.The results showed that the TYR activity,pheomelanin,eumelanin,and plasma colorimetry of melanin content of Lanping Black-boned Sheep were significantly higher than those of ordinary sheep in both regions（P<0.01）.Moreover,the alkaline-soluble melanin of Lanping Black-boned Sheep was significantly higher than that of Lanping ordinary sheep（P<0.01）,but there was no significant difference in total melanin content among the three sheep populations（P>0.05）.Notably,both total melanin and eumelanin contents were influenced by sheep sex,while eumelanin content was also affected by age,and these effects were significant（P<0.05）.2.The coding region of the WWOX gene in Lanping Black-boned Sheep was successfully obtained,which spans 1436 bp and encodes a protein consisting of 414amino acids.Based on protein structure prediction,the WWOX protein is hydrophilic and non-secretory,and predominantly located in the cytoplasm.This is the first report of the coding region of WWOX gene in Lanping Black-boned Sheep.3.Nine exons of the WWOX gene were successfully cloned from Lanping Black-boned Sheep,and a sequence comparison of the three populations revealed the presence of seven synonymous mutant loci with varying frequencies.Statistical analyses of gene and genotype frequencies of polymorphic loci unveiled significant differences in genotype frequencies between Lanping Black-boned Sheep and their common sheep counterparts from Lanping and Huize（P<0.05）,while allele frequencies did not display significant significantly（P>0.05）.Interestingly,neither the Exon2-C19G locus nor the Exon8-C140T mutation site in Lanping Black-boned Sheep adhered to Hardy-Weinberg equilibrium（P>0.05）.The TT genotype of Exon1-C102T was conspicuously absent in Lanping normal sheep and Huize common sheep,while the CC genotype was exclusively detected in Lanping normal sheep.The GG genotype was prevalent in both regions of common sheep at the locus Exon6-G72A.Furthermore,the loci Exon1-C102T and Exon2-C19G,Exon3-A41G,Exon8-C140T,Exon9-A45G,and Exon9-G96A exhibited high interlinkages between loci Exon3-A41G and Exon6-G72A,and Exon3-A41G and Exon6-G72A displayed tight interlinkages between loci Exon1-C102T and Exon2-C19G,Exon3-A41G,Exon8-C140T,Exon9-A45G,and Exon9-G96A in Lanping Black-boned Sheep.4.In the sheep population,the association analysis between melanin content and seven synonymous mutation loci of the WWOX gene was conducted,revealing that in Lanping Black-boned Sheep,the CC and CT genotypes of Exon1-C102T and Exon8-C140T,CC/GG of Exon2-C19G,GG genotype of Exon6-G72A,and Exon3-A41G,Exon9-A45G,and Exon9-G96A loci were significantly higher（P<0.05）than those in common sheep from both regions,for five indices including.The dominant haplotype in Lanping Black-boned Sheep was found to be CCGGCGG,whereas in Lanping normal sheep and Huize common sheep,the dominant haplotypes were CCGGCGG and CCAGCGG,respectively.5.Upon conducting whole-mount in situ hybridization of zebrafish,we observed that the expression of the WWOX gene was non-specific and widespread,resembling the expression pattern of an atypical pigment gene.The CRISPR-edited mutant showed a high level of confidence with a fit of 0.94 when compared to the reference sequence.The two g RNAs used at 36hpf and 48hpf produced a 67%indel,resulting in slightly more pigment deposition at 36hpf and significantly more melanin deposition at the yolk extension and tail of 48hpf than the wild type.Furthermore,the g RNA1-only edited gene at 72hpf exhibited a 42%indel,causing some alterations in the pigment phenotype.These results suggest that the WWOX gene may play a role in the regulation of melanin synthesis.6.The utilization of CRISPR/Cas9 gene editing technology facilitated the generation of WWOX^+/-mutants,which were analyzed via q RT-PCR to determine their impact on the expression of melanin-related genes.Strikingly,the transcriptional activity of TYR and TYRP1a genes was significantly upregulated（P<0.01）at both36h and 48h,while DCT was found to be moderately increased（P<0.05）.Notably,the expression of TYR in the control group at 72h was remarkably decreased（P<0.01）.These findings unveiled a negative regulatory role of WWOX gene on TYR,TYRP1a,and DCT.Hence,it is inferred that the WWOX gene exerts a closely knit relationship with the expression of melanin-related genes.To sum up,we report the first cloning of the WWOX gene in Lanping Black-boned Sheep,which revealed seven synonymous mutant loci,including Exon1-C102T,Exon2-C19G,Exon3-A41G,Exon6-G72A,Exon8-C140T,Exon9-A45G,and Exon9-G96A,all of which were significantly associated with melanin index.To explore the functional significance of WWOX gene,we employed a zebrafish model and generated CRISPR/Cas9 knockout mutants,which exhibited altered pigmentation phenotype.In addition,whole-mount in situ hybridization analysis showed that WWOX had extensive and non-specific expression pattern similar to that of atypical pigment gene.Further q RT-PCR analysis revealed that WWOX negatively regulates TYR,TYRP1a and DCT,suggesting a close relationship between WWOX and related melanin genes.Collectively,our findings provide new insights into the molecular mechanisms underlying melanin synthesis in Lanping Black-boned Sheep.