| Albinism of vertebrate body color is a common phenomenon in nature,the typical cave fish living in the dark environment in particular.Through laboratory feeding and observation of two types of typical cave fish collected in Libo county of Guizhou: Oreonectes daqikongensis and Oreonectes jiarongensis,it has been found that no change of body color happens to Oreonectes daqikongensis in the lab and that the body color of Oreonectes jiarongensis returns to black after 14 days’ feeding in the lab.This study takes Oreonectes daqikongensis and Oreonectes jiarongensis as research objects.Polymerase Chain Reaction(PCR)technology is applied to amplify the major genes(MC1R,OCA2 and TYR)of melanin synthesis and High-throughput sequencing technology to further study gene existence and gene expression in the skin of Oreonectes daqikongensis and two phenotypes of Oreonectes jiarongensis.The genes and pathways involved in the melanin synthesis were explored by comparing the differentially expressed genes in the transcriptomes of the two types Oreonectes.The exploration of the body color genes provides valuable information for subsequent functional gene analysis and adaptive evolution analysis of cave organisms.The main research contents are as follows:1.RNA-seq is applied and it is found that in the skin tissues of Oreonectes daqikongensis clean reads are 11.95 Mb,after further redundancy and assembly,the 66647 Unigenes are obtained.Albino of Oreonectes jiarongensis in the skin tissues clean reads are 23.56 Mb,after further redundancy and assembly,the 69568 Unigenes are obtained,and black phenotype of Oreonectes jiarongensis in the skin tissues clean reads are 24.06 Mb,after further redundancy and assembly,the 76564 Unigenes are obtained.KEGG and COG gene annotation finds that there is no significant difference in the functional classification of gene expression of the two Oreonectes.2.Related genes of melanin synthesis MC1 R,OCA2,MITF,TYR,TYRP1 and DCT don’t express,but L-tyrosine are express 13.78.The analysis of gene expressing content of two phenotype of Oreonectes jiarongensis in the skin shows 14533 genes of low expression.Themelanin synthesis related genes are expressed in two phenotype of Oreonectes jiarongensis,but this genes expressing content in black phenotype of Oreonectes jiarongensisits more than albino phenotype of Oreonectes jiarongensis.KEGG analysis shows: Many of genes expression contents up regulation of oxidative phosphorylation,tricarboxylic acid cycle,fatty acid synthesis and pentose phosphate pathway,however,many of genes expression contents down regulation of melanin synthesis pathway,MAPK signaling pathway,fat digestion and absorption and tyrosine metabolism in Oreonectes daqikongensis and two phenotypes of Oreonectes jiarongensis.This was consistent with the behavior that reduced sleep and increased foraging for a long time in Oreonectes daqikongensis and Oreonectes jiarongensis.Speculated that this phenomenon is relate to physiological regulation that increase the foraging time and reduced sleep time in cave environment with Oreonectes daqikongensis and Oreonectes jiarongensis.3.Accoding to RNA-seq,through PCR technology amplifies the major genes(MC1R)of melanin synthesis.The encoding region sequence of MC1 R is 937 bp and 988 bp of Oreonectes daqikongensis and Oreonectes jiarongensis.Sequence alignment of congeneric species(sibling species)and amino acid sequence are predicted.The research result shows: There is a deletion of nucleotide sequence(29bp)in the encoding region sequence of MC1 R of Oreonectes daqikongensis,leading to frameshift mutation,that is,termination codon TGA appears in the 84 th amino acid site.This mutation leads to loss in gene function and the melanin synthesis process to stop,thus causing the albinism of Oreonectes daqikongensis.No mutation site was found in MC1 R gene of Oreonectes jiarongensis.Meanwhile,amplifies the exon 21 and exon 24 of OCA2 and exon 4 of TYR genes validates Oreonectes daqikongensis and Oreonectes jiarongensis no mutation with Astyanax mexicanus.4.In the amplification of body color candidate gene in Oreonectes jiarongensis,no mutation site was found.All the genes involved in melanin synthesis are expressed in transcriptome analysis.Thus,the related genes of synthesis of melanin do not directly affect the change in body color of Oreonectes jiarongensis.But in the analysis ofneurohormone expression which mediates the body color,it is found that in albino phenotype Oreonectes jiarongensis,the expression of catecholamines,epinephrine and other neurohormones which gather pigment granules is higher than that of black phenotype Oreonectes jiarongensis and Oreonectes daqikongensis.Acetyl choline and melanocyte-stimulating hormone which make pigment granules diffuse and body color black,are not expressed in albino phenotype Oreonectes jiarongensis.Therefore,it is conjectured that the albinism of body color of Oreonectes jiarongensis is mainly caused by the mediation of neurohormone. |
