为探究喀斯特洞穴的代表物种——红点齿蟾蝌蚪(Oreolalax rhodostigmatus)白化的分子调控机制,采用Illumina HiSeq™4000对白化和黑色红点齿蟾蝌蚪皮肤组织进行了转录组测序,探寻基因表达差异,为红点齿蟾适应性进化提供理论依据.结果显示,转录组拼接组装后获得98 711个Unigene,总长度为96 459 902 bp,平均长度为977 bp;共预测3 389个完整开放阅读框(open reading frame,ORF),其中长度分布在0～200个氨基酸的ORF最多,占ORF总数的74.89％.Deseq2分析共筛选出269个差异表达基因(DEGs),其中上调基因136个,下调基因133个.GO功能注释发现,差异表达基因显著富集在细胞过程、代谢过程、结合、催化活性等通路上.KEGG(Kyoto Encyclopedia of Genes and Genomes)信号通路分析显示,差异基因被成功富集到175个信号通路上,主要包括代谢途径、脂肪酸合成、酪氨酸代谢、甘氨酸、丝氨酸和苏氨酸代谢等通路,筛选到酪氨酸代谢通路、黑色素合成通路、MAPK信号通路、WNT信号通路、PI3K-Akt信号通路与黑色素合成相关.通过荧光定量PCR验证转录组数据的可靠性.最后利用MISA查找到9 623个SSR位点,出现频率最高的为二碱基重复(79.87％).本实验结果可为红点齿蟾蝌蚪白化性状相关基因的挖掘与功能研究提供基础信息.
Transcriptome Analysis of Albinism in Tadpoles of Toad Oreolalax rhodostigmatus
In order to explore the molecular regulation mechanism of albino in toad Oreolalax rhodostigmatus,a representative species of karst caves,Illumina HiSeqTM 4000 technology was used to define the transcriptome of the skin tissue of albinism and black tadpoles of toad O.rhodostigmatus to explore the differences in gene expression and to provide theoretical basis for the adaptive evolution of the toad.The results showed that a total of 98 711 Unigenes were obtained by de novo splicing,and a total length was 96 459 902 bp,with an average length of 977 bp.A total of 3 389 complete open reading frames(ORFs)were predicted,with predominant ORFs with a length distribution among 0-200 aa,reaching a ratio of 74.89％among total ORFs.DESeq2 analysis identified 269 differentially expressed genes(DEGs)between the two groups of toad tadpoles,with 136 up-regulated genes and 133 down-regulated genes.Gene ontology functional enrichment analysis revealed that many of these genes encoded proteins involved in cellular process,metabolic process,binding and catalytic activity.KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis showed that these DEGs were significantly enriched in 175 signaling pathways,including metabolic pathways,fatty acid metabolism,tyrosine metabolism,glycine,serine and threonine metabolism,and among them,tyrosine metabolic pathway,melanin synthesis pathway,MAPK signaling pathway,WNT signaling pathway and PI3K-Akt signaling pathway were associated with melanin synthesis.The validity of transcriptome data was verified by fluorescence quantitative PCR.In addition,9 623 SSR loci were detected,with the maximal frequency of dinucleotide repeats(79.97％).The findings will provide basic information for the exploration and functional research of genes related to the albinism trait of O.rhodostigmatus tadpoles.
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