| Strongylocentrotus intermedius belongs to the cold water type echinoderms.It was introduced from Japan by Dalian Ocean University in 1989.Because of its fast growth,gonads rich in unsaturated fatty acids and high economic value,it has become one of the main species of sea urchin culture in my country[1].However,in recent years,the seawater temperature in the Yellow Sea and Bohai Sea has increased[2],which has seriously restricted the development of the sea urchin industry.In this study,S.intermedius with a body weight of 26.72～33.72g,a shell diameter of33.36～46.25mm,and a shell height of 15.27～24.32mm was used as the research object.Activity and heat shock gene expression,and screen the differential genes and differential proteins of S.intermedius using the whole transcriptome and TMT proteome,and preliminarily explore the molecular mechanism of S.intermedius heat tolerance.The main findings obtained are as follows:1).Determination of heat resistance.In this study,the S.intermedius heat resistance index（UTT）,critical high temperature value and survival analysis were determined and analyzed.The results showed that S.intermedius began to die under temperature stress at 25℃;the UTT value of heat resistance index ranged from 115 to143℃/h,and the critical high temperature value（CTmax）ranged from 27.5 to 29℃.High temperature tolerance results for red and white tube feet S.intermedius show:The heat resistance index（UTT）,critical high temperature value（CT max）,estimated mean and estimated median survival time of red tube feet S.intermedius were significantly higher than those of white tube feet S.intermedius,The above results indicated that the temperature tolerance of red tube feet S.intermedius was significantly higher than that of white tube feet S.intermedius.2).Antioxidant enzyme and heat shock gene expression validation.To further verify the heat resistance of red and white tube feet S.intermedius,the activities of five antioxidant enzymes and the expression of four heat shock genes in the reference genome were tested.The immunoenzyme activity of S.intermedius under temperature stress showed that the activities of the five enzymes all increased when the temperature increased.Among them,glutathione peroxidase（GSH-PX）had the highest activity,with an activity range of 249-543 U/m L;peroxidase（POD）and superoxide dismutase（SOD）had the lowest activity,with their activity ranges respectively were 0.34～3.42U/m L and 7.29～35.20U/m L.The activities of five antioxidant enzymes in white tube feet S.intermedius were higher than those in red tube feet S.intermedius in the experimental and control groups.The results of heat shock gene expression showed that under temperature stress,the relative expression levels of the four heat shock genes in S.intermedius ranged from 1.18 to 21.71,and hsp70A,hsp70B,hsp60 and hsp90 were significantly increased（P<0.05）.,the expression level of heat shock genes of red tube feet S.intermedius under high temperature stress was significantly higher than that of white tube feet S.intermedius（P<0.05）.The above results indicate that red tube feet S.intermedius has higher temperature tolerance than white tube feet S.intermedius.3).Transcriptome-based differential gene expression analysis of S.intermedius.Through whole transcriptome sequencing of S.intermedius under temperature stress,1852,35,1254 and 71 differentially expressed mRNAs,miRNAs,lncRNAs and circ RNAs were screened from 12 sequencing libraries,respectively.Genes that might be involved in the regulation of heat resistance were screened by reference genome annotation and NCBI database.The results showed that the significantly differentially expressed mRNAs included PP4C,CYP2J3,PYR1,CP2U1 and SRCR1,etc.,which were confirmed by RT-q PCR to be consistent with the sequencing results.Apoptosis is closely related;significantly differentially expressed miRNAs include spu-miR-133,spu-miR-92b-3p and novel＿128,etc.,it is speculated that these miRNAs may play an important role in S.intermedius response to temperature stress;significantly differentially expressed lncRNAs include TCONS＿00090931,TCONS＿00065041and TCONS＿00085394,etc.,were annotated as LIMK1,SPRR3 and P2RX7 genes respectively through target genes,and the annotation indicated that these genes were closely related to cellular immunity,cell proliferation and apoptosis;TPM boxplots showed that Circ RNAs had no effect on S.intermedius in response to high temperature stress.significant impact.KEGG analysis of differential genes showed that endoplasmic reticulum protein processing pathway,glycine,serine and threonine metabolism,and Notch signaling pathway were significantly enriched,which may be involved in the regulation of S.intermedius heat tolerance.Competitive endogenous RNA（ceRNA）regulatory relationships were constructed,in red tube feet S.intermedius,such as TCONS＿00132936-novel＿128-（PP4C,CND3,RDH8,PYR1 and CP2U1）,TCONS＿00179583-spu-miR-133-CLCA4A;In white tube feet S.intermedius,such as TCONS＿00064455-spu-miR-92b-3p-（SRCR1,EGG1 and SISin01G004090.1）.4).Proteome-based analysis of differential protein identification of S.intermedius.S.intermedius proteome sequencing under temperature stress showed that the functions of S.intermedius were mainly enriched in protein binding;post-translational modification,protein turnover,chaperone;metal ion binding and other processes.Among them,significant enrichment in red tube feet S.intermedius included protein dephosphorylation,regulation of phosphoprotein phosphatase activity and ascorbic acid and aldose metabolism;significant enrichment in white tube feet S.intermedius included lipid transporter activity and protein processing in the endoplasmic reticulum.Proteins that may be related to the regulation of heat resistance were screened by protein annotation and NCBI database.The screening results showed that the significantly different proteins included heat shock proteins,calcium ion-binding proteins,eukaryotic porin/Tom40,cell division cyclins,ubiquitin ligases,etc.It was speculated that the above proteins were related to the thermal resistance of S.intermedius. |
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