摘要：

胴体生长发育是影响山羊养殖效益的重要评价指标,旨在通过RNA-Seq技术挖掘不同性别贵州白山羊生长发育的关键候选基因,为贵州白山羊生长发育研究提供理论依据,以同等饲养水平条件下贵州白山羊为研究对象,测定2周龄不同性别贵州白山羊的屠宰性能指标,通过背最长肌转录组分析,筛选差异表达基因,分析相关基因的信号通路,最后通过RT-qPCR对筛选的基因进行验证。结果表明,测序得到的Raw reads进行过滤后,6个样本共得到78.99 Gb Clean Data,单个样本获得Clean reads 在83 030 104~95 739 024条,各样本与参考基因组的比对效率在93.75%~94.79%,共获得25 089个表达基因,筛选差异表达基因共获得1 077个差异基因,其中194个为新发现转录本基因,发现的差异基因中563个在公羊背最长肌中表达显著上调,514个表达显著下调。对获得的1 077个差异基因进行GO功能富集分析,其中587个为显著富集(Q-value≥0.05),主要分为三大类35个亚类。KEGG信号通路分析发现,这些差异基因共参与243条信号通路,其中参与PI3K/AKT信号通路相关的基因最为富集,共注释到32个基因,其中17个注释基因显著上调,1个显著下调,且在该通路中COL4A1和COL4A2基因编码蛋白共表达估值最高,ITGAV基因编码蛋白互作最为丰富。筛选出6个与贵州白山羊背最长肌生长发育相关的差异表达基因进行验证,其中FHL3、WFIKKN2、SOX6基因在公羊背最长肌中为上调基因,QSOX2、MYH2、LAP基因为下调基因,RT-qPCR结果显示,这6个候选基因的mRNA表达趋势与转录组测序结果一致,且差异均较为显著(P<0.05),表明测序结果可靠。基于RNA-Seq技术筛选出不同性别贵州白山羊背最长肌的差异表达基因1 077个,其中194个为新发掘转录本基因,筛选的差异表达基因及PI3K/AKT信号通路与贵州白山羊背最长肌生长发育相关。

关键词: 贵州白山羊, RNA-Seq技术, 不同性别, 生长发育, 差异表达基因

Abstract:

Carcass muscle growth and development is an important evaluation index that affects the breeding efficiency of goats.The aim of this study was to analyze key candidate genes for muscle growth and development of Guizhou white goats with different genders by RNA-Seq,and provide new reference information for the research on the muscle growth and development of Guizhou white goat.We collected the longissimus dorsi muscle to extract RNA and determined the slaughter performance of 6 Guizhou white goats with different genders,which were two years old and were feeded in same level.Then screened differentially expressed genes,analyzed the signal pathway of related genes.Meanwhile,RT-qPCR was used to verify the screened differentially expressed genes.The results showed that the raw reads which obtained by sequencing were filtered,a total of 78.99 Gb Clean Data were obtained in six samples.Each single sample was obtained Clean reads between 83 030 104 and 95 739 024,and the comparison efficiency with the reference genome was between 93.75%-94.79%,a total of 25 089 transcripts were obtained,of which 1 077 were significant differentially expressed genes,and 194 were new transcripts.Among them,563 were up-regulated and 514 were down-regulated in longissimus doris tissue of male sheep.GO functional enrichment analysis was performed on 1 077 differentially expressed mRNAs,which 587 were significantly enriched(Q-value≥0.05) and concentrated in 35 groups of three major categories.KEGG signaling pathway analysis revealed that annotated differential genes participated in 243 signaling pathways,among which the PI3K/AKT signaling pathway were the most enriched,including 32 genes,among them,17 genes were significantly up-regulated and 1 gene was down-regulated,meanwhile,the coexpression score of COL4A1 and COL4A2 genes were the highest in this pathway,the ITGAV protein interaction with other proteins was the most abundant.6 genes that may be closely related to muscle growth and development in white goat were screened out,among which FHL3,WFIKKN2 and SOX6 genes were up-regulated in male longissimus doris tissue,QSOX2,MYH2 and LAP genes were down-regulated.RT-qPCR showed that the expression trend of the 6 candidate genes were consistent with high-throughput sequencing results,and the expression levels differences of these genes were significant,which showed the sequencing results were reliable.Totally,1 077 differentially expressed mRNAs,which 194 were new transcripts were obtained of longissimus doris tissue in Guizhou white goat with different genders by RNA-Seq technology,the screened differentially expressed genes and PI3K/AKT signaling pathway were related to the growth and development of longissimus dorsi muscle of Guizhou white goats.

Key words: Guizhou white goat, RNA-Seq technology, Different genders, Growth and development, Differentially expressed genes