基于RNA-Seq鉴定黑羽番鸭肉质风味差异的候选基因

Identification of candidate genes related to meat flavor in black Muscovy duck based on RNA-Seq

为挖掘黑羽番鸭肉质风味相关候选基因,通过对黑羽番鸭胸肌、腿肌中挥发性风味物质进行测定,并通过Illumina HiSeq2500高通量测序进行转录组对比分析,结合参考基因组对所获得的序列进行序列比对、基因注释和差异表达等分析,筛选出差异表达基因并进行GO富集分析。通过荧光定量PCR(Real-time PCR,qRT-PCR)方法检测4个差异候选基因表达水平,验证测序结果的可靠性。结果表明,黑羽番鸭胸肌中挥发性风味物质辛醛、2,3-辛二酮含量显著高于腿肌,3-羟基-2-丁酮含量显著低于腿肌(P<0.05)。通过参考基因组比对和差异表达分析,初步获得614个差异表达基因,171个差异基因在黑羽番鸭腿肌组织上调表达,443个差异基因在黑羽番鸭腿肌组织下调表达。结合GO分析和KEGG富集分析,最终获得了20个候选功能基因,它们分别参与机体氨基酸形成、糖代谢以及脂肪代谢等生物过程,其中10个差异候选基因在黑羽番鸭胸肌中参与肌内脂肪代谢过程,这些基因可能通过形成肌内挥发性风味物质进而影响肌肉风味。qRT-PCR验证结果表明,筛选出的差异候选基因表达趋势与转录组测序中表达的趋势相似,说明测序结果可靠。在黑羽番鸭胸肌、腿肌中挥发性风味物质对比基础上,结合RNA-Seq技术对黑羽番鸭肉质风味前体物质特异性形成机制进行综合分析,筛选出ACOT11、APOA1、FABP3等脂肪代谢相关的差异基因可能与黑羽番鸭胸、腿肌肉质风味形成相关。

To explore the candidate genes related to meat flavor in black Muscovy duck,volatile flavor compounds in breast and leg muscles of black Muscovy duck were determined,and transcriptome analysis was performed by Illumina HiSeq2500 high-throughput sequencing.Based on reference genome,the obtained sequences were analyzed by sequence alignment,gene annotation and differential expression analysis,and differentially expressed genes were screened out and GO enrichment analysis was performed.Moreover,to verify the sequencing results,qRT-PCR was used to detect the expression levels of four differential candidate genes.Results showed that the contents of octanal and 2,3-octanedione in breast muscle were significantly higher than those in leg muscle,and the content of 3-hydroxy-2-butanone in breast muscle was significantly lower than that in leg muscle(P<0.05).Through genome comparison and differential expression analysis,614 differentially expressed genes were preliminarily obtained,171 differentially expressed genes were up-regulated and 443 differentially expressed genes were down-regulated in leg muscles of black Muscovy duck.By GO and KEGG enrichment analysis,20 functional candidate genes were finally obtained.These candidate genes were involved in biological processes,such as amino acid formation,glucose metabolism and fat metabolism.Among them,ten differentially expressed candidate genes were involved in fat metabolism in breast muscle of black Muscovy duck,and these genes may affect muscle flavor by forming intramuscular volatile flavor compounds.Results of qRT-PCR verification showed that the expression trends of screened differentially expressed candidate genes were similar to those in transcriptome sequencing,indicating that the sequencing results were reliable.Based on the comparation of volatile flavor compounds in breast and leg muscles of black Muscovy duck,the specific formation mechanism of meat flavor precursors in black Muscovy duck was comprehensively analyzed,differentially expressed genes related to fat metabolism such as ACOT11,APOA1,FABP3 may be related to the formation of meat flavor in breast and leg muscles of black Muscovy duck.