福清山羊与努比亚黑山羊背最长肌比较转录组分析

Transcriptome Analysis of Differentially Gene Expression Associated with longissimus doris Tissue in Fuqing Goat and Nubian Black Goat

【目的】研究福清山羊与努比亚黑山羊背最长肌组织转录组差异表达水平,为地方山羊品种肉质、生长性状的遗传机制和遗传改良提供理论基础。【方法】分别测定周岁内羯羊育肥的福清山羊与努比亚黑山羊日增重(ADG)以及2个品种周岁背最长肌样品的肌内脂肪含量(inter-muscular fat,IMF);同时利用转录组测序方法对2个品种周岁背最长肌组织进行高通量测序,筛选品种间的差异表达基因(differentially expressed genes,DEGs),并对DEGs功能进行注释和测序结果的荧光定量PCR(quantitative real-time PCR,qRT-PCR)验证。【结果】在羯羊育肥条件下,福清山羊周岁内ADG为65.6g·d-1,极显著低于努比亚黑山羊的127.4g·d-1(Sig.=0.000);而福清山羊周岁背最长肌IMF含量为3.69%,极显著高于努比亚黑山羊的1.83%(Sig.=0.003)。2个品种6个样品的背最长肌转录组测序共得到44.76Gb Clean Data,各样品的Clean reads与参考基因组(山羊)的比对效率在84.65%-86.17%之间。DESeq分析发现了努比亚黑山羊和福清山羊背最长肌的DEGs 608个,其中上调基因61个,下调基因547个。608个DEGs中的518个基因能够被GO(gene ontology)数据库注释,148个DEGs能够被COG(Cluster of Orthologous Groups of proteins)数据库注释,418个DEGs能够被KEGG(kyoto encyclopedia of genes and genomes)数据库注释。KEGG通路分析表明DEGs共富集到222条信号通路中,44条通路显著富集。经过文献检索和对筛选通路相关基因功能的分析,初步判断可能与羊肉质和生长性状相关的通路包括肌动蛋白细胞骨架调节(Regulation of actin cytoskeleton)、Jak-STAT信号转导通路{Janus kinase/signal transducer and activator of transcription(Jak-STAT)signaling pathway}、MAPK信号转导信号通路{mitogen-activated protein kinase(MAPK)signaling pathway}和Ⅰ型糖尿病通路(TypeⅠdiabetes mellitus);IGF1(Insulin-like growth factor 1,类胰岛素一号增长因子)、ACSL5(Long-chain fatty acyl-CoA synthetases 5,长链酯酰辅酶A合成酶5)、PCK2(phosphoenolpyruvate carboxykinase 2,磷酸烯醇丙酮酸羧激酶2)、PPARGC1A(Hepatic peroxisome proliferator-activated receptor gamma,coactivator 1 alpha,过氧化物酶体增殖激活受体γ共激活因子1α)、JAK2(Janus Kinase 2,Janus激酶2基因)、STAT4(signal transducer and activator of transcription 4,信号转导子和转录激活子4)、IRF8(interferon regulatory factor 8,干扰素调节因子8)、MAP4K1(mitogen-activated protein kinase kinase kinase kinase 1,有丝分裂原激活蛋白激激激激酶1)等DEGs可作为控制福清山羊肉质、生长性能的候选基因。进一步与参考基因组序列比对分析,共发掘707个新基因(转录本),其中15个基因(转录本)为2个品种的DEGs。经qRT-PCR验证,所选基因(转录本)表达变化模式与转录组测序结果一致,表明测序结果可靠。【结论】在转录组水平上筛选出了福清山羊和努比亚黑山羊周岁羯羊背最长肌组织的608个DEGs,发掘了707个新基因(转录本),初步认为肌动蛋白细胞骨架调节等4个信号通路在山羊肉质形成和生长发育过程中发挥了重要作用,为进一步探索山羊骨骼肌生长发育和IMF沉积的相关机理提供参考依据。

【Objective】The aim of this study was to analyze the transcriptome differentially genes expression of longissimus doris tissue in Fuqing goat and Nubian Black goat.【Method】Two goat breeds,including Fuqing goat and Nubian Black goat,were used as experimental animal,and fatten of castrated ram were used as experimental samples.We determined the samples’ADG in 12months,and IMF content in longissimus doris tissue at the age of 12 months.Transcriptome sequencing of longissimus doris tissue in Fuqing goat and Nubian Black goat were performed by using the Illumina HiSeqTM 2500 platform with 3 biological replicates per goat breed,and verified by quantitative real-time PCR(qRT-PCR).Differentially expressed genes(DEGs)were selected and enriched based on GO and COG and KEGG database.【Result】By fatten of castrated ram,the ADG of Nubiya Black goats in 12 months was higher that of Fuqing goats(Sig.=0.000).But,the IMF content in longissimus doris tissue of Fuqing goats at age of 12 months was higher than that of Nubiya Black goat(Sig.=0.003).A total of 44.76 Gb clean data were obtained in six samples.We found 608 DEGs between Fuqing goat and Nubian Black goat,including 61 DEGs up-regulated genes and 547 DEGs down-regulated genes.Moreover518 DEGs and 148 DEGs and 418 DEGs were enriched by GO and COG and KEGG database,respectively.KEGG pathway analysis showed that DEGs annotated to 222 metabolic pathways,and 44 pathways were enriched significantly,such as regulation of actin cytoskeleton,and Jak-STAT signaling pathway,and MAPK signaling pathway and TypeⅠdiabetes mellitus associated with meat quality and growth traits in goat.The results showed that 8 DEGs(IGF1,ACSL5,PCK2,PPARGC1A,JAK2,STAT4,IRF8 and MAP4K1)might relate to meat quality and growth traits of goat were screened by GO functional enrichment and KEGG Pathway analysis.In addition,707 new genes or transcripts were found by BLAST,including 15 DEGs between two breeds.By qRT-PCR verification,the pattern of selected genes was consistent with the results of transcriptome sequencing,which showed the sequencing results were reliable.【Conclusion】Totally,608 DEGs and 707 new genes of longissimus doris tissue in Fuqing goat and Nubian Black goat were screened by transcriptional analysis,and revealed that four pathways might play an important role in goat meat quality and growth traits.