基于转录组测序对剑白香猪黑色素生成相关基因的筛选与分析

Screening and analysis of genes related to melanin production in Jianbaixiang pig based on transcriptome sequencing

毛色作为猪的品种特征,有着重要的研究意义。本研究为筛选剑白香猪黑色素生成相关基因,通过转录组测序技术对贵州剑白香猪黑色被毛皮肤和白色被毛皮肤进行转录组测序,并运用qRT-PCR和免疫组化技术验证测序结果。结果识别差异表达基因1230个,其中相较于白色被毛皮肤组织而言,黑色被毛皮肤组织中上调基因有738个,下调基因有492个,包括与黑色素合成相关基因,如酪氨酸酶基因(tyrosinase,TYR)、酪氨酸相关蛋白1基因(tyrosinase-related protein1,TYRP1)、多巴色素异构酶(dopachrometautomerase,DCT)、黑色素A基因(melan-A,MLANA)等,且这些基因在剑白香猪黑色被毛皮肤中的表达量显著高于白色被毛皮肤。基因本体(gene ontology,GO)分析发现共有1070个差异表达基因显著富集于9个GO term,包括蛋白降解(proteolysis)、生物调节(regulation of biological quality)、细胞连接复合体(extracellular matrix organization)、细胞质(cytoplasm)、细胞核(nucleus)及ATP结合(ATP binding)等。KEGG通路分析表明酪氨酸代谢和黑色素合成通路显著富集,黑色素合成通路和酪氨酸代谢通路与剑白香猪毛色形成密切相关,参与黑色素合成通路的有TYRP1、TYR、DCT、WNT10B、WNT16、WNT3、WNT11、CREB3L2等11个差异表达基因,参与酪氨酸代谢通路的基因有TYRP1、TYR、DCT、AOX1。经qRT-PCR对TYR、TYRP1、DCT、SLC45A2、MLANA等部分基因验证,基因表达变化情况与转录组测序结果一致;进一步通过组织化学染色方法对剑白香猪皮肤中MITF和TYR基因的蛋白表达水平进行分析,MITF和TYR蛋白在香猪黑色被毛皮肤中的表达量均高于白色被毛皮肤,表明测序结果准确。通过SNP分析发现在剑白香猪白色被毛皮肤组织测序结果中,SLC45A2和TYRP1基因存在错义突变的SNP,SLC45A2有2个错义突变的SNP位点:Exon1-g.229G>A和Exon1-g.13499A>G;TYRP1有1个错义突变的SNP位点:Exon7-g.17261A>G。研究结果提示,剑白香猪中TYRP1、TYR、DCT、SLC45A2、MLANA、PMEL、WNT10B、WNT16、WNT3、WNT11、CREB3L2等基因的下调可能是造成剑白香猪白色被毛的原因之一,揭示酪氨酸代谢和黑色素合成信号通路在剑白香猪毛色表型形成中发挥了重要作用,SLC24A5和TYRP1基因的突变也可能影响剑白香猪的毛色。本研究为剑白香猪不同毛色皮肤组织基因表达谱提供了新的信息,为探究猪毛色性状的遗传机制及分子标记育种提供了基础材料。

Coat color,as a breed characteristic of pigs,has important research significance.This study aims to screen the genes related to melanin production in Jianbaixiang pig.Transcriptome sequencing was performed on the black coat skin and white coat skin of Guizhou Jianbaixiang pig by transcriptome sequencing technology,the qRT-PCR and immunohistochemistry technology was used to verify the sequencing results.The results identified 1230differentially expressed genes.Compared with white coat skin tissues,there were 738up-regulated genes and 492down-regulated genes in black coat skin tissues,including genes related to melanin synthesis such as casein gene,tyrosinase(tyrosinase,TYR)gene,tyrosine-related protein 1(tyrosinase-related protein 1,TYRP1)gene,dopachrome isomerase(dopachrometautomerase,DCT)gene,melanin A(melanA,MLANA)gene,etc.The expression level of these genes in the black coat skin of Jianbaixiang pig was significantly higher than that in the white coat skin.Gene Ontology(GO)analysis found that a total of 1070differentially expressed genes were significantly enriched in 9GO terms,including protein degradation(proteolysis),biological regulation(regulation of biological quality),and cell junction complex(extracellular matrix organization),cytoplasm,nucleus;ATP binding,etc.KEGG pathway analysis showed that tyrosine metabolism and melanin synthesis pathway pathways were significantly enriched,melanin synthesis pathway and tyrosine metabolism pathway were closely related to Jianbaixiang pig hair color formation.There were 11differentially expressed genes involved in the melanin synthesis pathway,including TYRP1,TYR,DCT,WNT10B,WNT16,WNT3,WNT11,CREB3L2,etc.The genes involved in the tyrosine metabolism pathway were TYRP1,TYR,DCT,AOX1.The results were validated by qRT-PCR on some genes such as TYR,TYRP1,DCT,SLC45A2,MLANA,the gene expression changes were consistent with the results of transcriptome sequencing.The protein expression of MITF and TYR genes in Jianbaixiang pig skin was further analyzed by histochemical staining method analysis,the results showed that the expression levels of MITF and TYR protein in the black coat skin were higher than those in the white coat skin,indicating that the sequencing results were accurate.SNP analysis of the sequencing results of the white coat skin tissue of Jianbaixiang pig found that SLC45A2and TYRP1genes had missense mutant SNPs,and SLC45A2had two missense mutant SNP sites:Exon1-g.229G>A and Exon1-g.13499A>G;TYRP1had a missense mutation SNP site:Exon7-g.17261A>G.The results suggested that the down-regulation of genes such as TYRP1,TYR,DCT,SLC45A2,MLANA,PMEL,WNT10B,WNT16,WNT3,WNT11,CREB3L2in Jianbaixiang pig may be one of the reasons for the white hair of Jianbaixiang pig,revealing tyrosine acid metabolism and melanin synthesis signal pathways play an important role in the formation of Jianbaixiang pig’s coat color phenotype.Mutations in SLC24A5and TYRP1genes may also affect Jianbaixiang pig’s coat color.This study provided new information for the gene expression profile of different coat color skin tissues of Jianbaixiang pig,and provided basic materials for exploring the genetic mechanism of pig coat color traits and molecular marker breeding.