Transcriptome Analysis And Research On The Related Function Gene In Response To Thermal Stress In Sinonovacula Constricta

Temperature is one of the major environmental stressors affecting the basic physiological processes in marine organism,such as growth,development.Global warming and increasing frequency of extreme weather causes increases in ocean temperature.The intertidal species are vulnerable to extreme environmental factors challenge during tidal changes and seasonal variability.The razor clam,Sinonovacula constricta,as an eurythermic marine economically bivalve,it is widely distributed in the estuarine and intertidal region.Unlike other intertidal mollusks,it cannot escape from environmental stress because of its limited range of activities.So,razor clams may have acquired adaptive mechanisms to defense environmental pressure in evolution.We speculated that it may be more resistant to higher water temperatures.However,high temperature is still one of the important environmental factors that affect its growth and induce diseases.Therefore,study the high temperature adaptability of clam and screen heat-resistant genes will contribute to understanding the molecular adaptive mechanism responding to heat stress and provide candidate genes for genetic breeding of the clams with high temperature resistant traits.In aquatic organisms,gills,as a primary respiration and metabolism organ,can directly contact with water and quickly response to temperature change,and hepatopancreas,as a critical organ for metabolism and immunoregulation is vulnerable to environmental stress.Therefore,to provide a reference data for further significant insights into the high temperature tolerance mechanism of razor clam,we performed transcriptome profiling of gills and hepatopancreas exposed to acute heat condition,and screen heat-resistant and functions verified of DEGs.1.The transcriptomic profiling of gills and hepatopancreas in S.constricta collected from both control(22 ℃)and treatment groups(32 ℃)for 24 h were investigated with Illumina sequencing.A total of 556 and 786 differentially expressed genes(DEGs)were obtained in gills and hepatopancreas,respectively.Among this,311 and 245 DEGs were up-regulated and down-regulated in gills,and 456 and 330 DEGs were up-regulated and down-regulated in hepatopancreas of treatment group compared to control group.GO and KEGG analysis showed that these DEGs were enriched in protein homeostasis,metabolisms,immune response,antioxidant response and cytoskeletal organization processes.To validate the RNA-seq data,seventeen heat response-relevant genes were selected for qRT-PCR analysis.In overall,the expression pattern of DEGs detected by qRT-PCR was highly similar to that identified by RNA-seq.Then,the expression profile of four significantly different genes in five normal adult tissues and the domain structures of genes were determined.The results showed that these genes have been similar domains and were quite conserved in different species.The highest expression of HSP70,GRP94,DNAJB4 and CYP1A2 were detected in mantle,foot and hepatopancreas,respectively.2.qRT-PCR technology was used to analyze the expression levels of three types temperature response candidate genes,include molecular chaperone genes(HSP70,HSF1、GRP94,PDIA6,CALR),immune related genes(CTL,CTSL),and apoptosis genes(caspase-3,p53)based on transcriptomic analysis in gills and hepatopancreas under different acute high temperature conditions(30 ℃,32 ℃,34 ℃).The results showed that the expression of molecular chaperone genes was significantly up-regulated at 4 h under thermal stress,and was positively correlated with temperature.Meanwhile,gills responded earlier than the hepatopancreas.The m RNA expression of immune responses and apoptotic genes exhibited an increase and then decrease in both tissues with the extension of stress time.In conclusion,the regulation of these genes was played significant roles to maintain basic homeostasis in S.constricta under heat stress.The immune response genes were more significant expression in hepatopancreas.3.The ScPDIA6 gene verification result showed that the open reading frame(ORF)is1331 bp which encoded 436 ammino acids(aa).PDIA6 contained a signal peptide composed of 18 amino acids and two thioredoxin domains.The phylogenetic tree analysis results ScPDIA6 has high homology with shellfish.The tissues expression profile results showed that the expression of PDIA6 was the highest in the mantle.The expression level of PDIA6 was significantly up-regulated in gill,hepatopancreas and heart after thermal stress(P<0.05).The PDIA6 m RNA was positively correlated with temperature in mantle and gill,and exhibited an increase and then decrease in both tissues with the extension of stress time.In addition,the gill was more sensitive than the mantle under thermal stress.The prokaryotic expression results showed that the optimal expression temperature of pET-32a-PDIA6 protein was 28 ℃,and the target protein was mainly expressed in the form of inclusion bodies.4.The verification result showed that the ORF of ScCALR gene is 1331 bp which encoded 406 ammino acids.It is a hydrophilic protein endoplasmic reticulum protein with no transmembrane structure,which including a signal peptide composed of 16 amino acids and two thioredoxin domains.The phylogenetic tree analysis of ScCALR showed it has high homology with shellfish.The qRT-PCR results showed that CALR was expressed in all tissues and highest expression in the kidney.The expression level of CALR was significantly up-regulated in gill,hepatopancreas and heart after thermal stress(P<0.05).The expression level of CALR was significant up-regulated,and increased first and then decreased with the temperature.The prokaryotic expression results showed that the optimal expression temperature of pET-32a-CALR protein was 37 ℃,and the target protein was mainly expressed of soluble protein.