基于转录组分析温度诱导黑斑原(Glyptosternummaculatum)选择性剪接的变化

Temperature Stress Induced Alternative Splicing in Glyptosternum maculatum Based on Transcriptome Analysis

将采自多雄藏布支流(N29°27’58.72",E 86°54’36.06")的90尾雌性黑斑原(Glyptosternum maculatum)分为3组,每组30尾:A组为对照组,水温为12℃;B组以2℃/h从12℃下降到4℃;C组以2℃/h上升,达到24℃时,黑斑原濒死时开始实验,利用转录组数据分析黑斑原在温度诱导后的选择性剪接的变化。结果表明:高温组(24℃)、对照组(12℃)和低温组(4℃)处理后,黑斑原肝脏中选择性剪接事件存在互斥外显子(MXE)和外显子跳跃(SE)两种类型;高温和低温处理后选择性剪接事件和基因均明显减少,高温组和低温组分别比对照组减少了16.39%、16.35%和5.17%、6.21%;与对照相比,24℃和4℃分别有75和32个特有基因高表达。GO分析显示,在24℃时分别有190、88和318个基因富集到生物学过程、细胞定位和分子功能,而4℃分别有101、50和210个基因富集到这3个GO分类中。KEGG分析显示,83和9通路分别显著富集在24℃和4℃,其中通路内质网蛋白质加工和嘌呤代谢在高温组和低温组共有,2个通路中显著差异基因为xdh、papss2、impdh1、polr3h、dnajc1、derl2和dnajc10,其中dnajc1基因在高温组和低温组均差异显著。dnajc1基因属hsp40亚家族成员,生物信息学分析发现dnajc1基因在4℃和12℃中均为1个剪接体,高温处理后产生了2个剪接体,说明dnajc1基因在黑斑原遭遇温度诱导后肝脏通过产生选择性剪接体来保护细胞内的稳态。本研究首次研究了黑斑原应对温度反应的选择性剪接,为了解鱼类应对非生物应激的分子机制提供理论基础。

In this study,90 individuals of Glyptosternum maculatum collected from the tributary of Doxiong Tsangpo(N29°27’58.72",E86°54’36.06")were divided into three groups:group A as the control group at water temperature of 12℃;in the group B,the water temperature dropped from 12℃to 4℃at 2℃/h,and group C the water temperature increased by 2℃/h,and at 24℃.Then the changes in alternative splicing were analyzed in the Glyptosternum maculatum induced by temperature by the transcriptome data(existing RNA-Seq datasets).It was found that there were only two type alternative splicing events including mutually exclusive exon(MXE),and exon skipping(SE)in the liver of G.maculatum in the three groups,the alternative splicing events after treatment by 24℃and4℃obviously decrease in group C and group B compared to the control group in 24℃treatment,with the thermal stress induced decrease by 16.39%in alternative splicing events and decrease by 16.35%in alternative splicing genes,in group B,decrease by 5.17%in alternative splicing events and decrease by 6.21%in alternative splicing genes in the low temperature stress.There were 75 and 32unique genes which were high expressed in 24℃treatment and 4℃treatment compared to 12℃.GO analysis showed that there were190,88 and 318 genes were enriched in biological processes,cell composition and molecular function in 24℃treatment,and 101,50and 210 genes in 4℃treatment.KEGG analysis showed that 83 and 9 pathways were enriched in 24℃treatment and 4℃treatment,respectively,in which protein processing in endoplasmic reticulum pathway and purine metabolism pathway were found at both high-heat and low-heat.xdh,papss2,impdh1,polr3h,dnajc1,derl2 and dnajc10 genes were shown to be significant difference both in the two pathways,in which dnajc1 gene was significant difference both in high-heat and low-heat.The bioinformatics analysis showed that the gene was a member of the hsp40 subfamily,and that there was 1 alternative splicing transcript at 4℃and 12℃treatment,while there were two alternative splicing transcripts at 24℃treatment,illustrating that dnajc1 gene in liver might protect homeostasis in the cell though alternative splicing when G.maculatum encountered heat stress.The finding is the first study of alternative splicing in response to heat stress in G.maculatum,and provides insights into the molecular mechanisms of responses to the abiotic stress.