Preliminary Study About Effects Of Starvationon Cold Tolerance Capacity In Large Yellow Croaker(Larmichthys Crocea)

This study is conducted to investigate the effects of starvation on cold tolerance ability in large yellow croaker(Larmichthys crocea).By detecting the differences of survival rate,blood physiological and biochemical indexes,fatty acid composition,and the expression of cold tolerance related gene between starvation group and control group,it is aimed to investigate the mechanisms of cold tolerance under starvation.The results are as follows:In this study,the results shows that when the temperature of seawater dropped to 7 °C,the cumulative mortality in five days of starvation group is 27%,while the cumulative mortality of control group is 58%.The blood physiological and biochemical indexes,including red blood cell count,hemoglobin,white blood cell count,cholesterol and triglyceride levels,are investigated.Results showed,the red blood cell count increased slightly in the stage of starvation,whereas the hemoglobin levels decreased slightly.In the cold stress stage,red blood cell count and hemoglobin level were parabolic relationship with temperature,but the levels of starvation group were higher than control group.White blood count of control group declined in the whole stage,and the count of starvation group declined rapidly at starvation stage and then maintained at low level.Cholesterol level in the cold stress stage was not significant between starvation and control group.The level of serum triglyceride declined significantly in the starvation stage,and the level showed a downward trend after first rise in cold stress stage,while the level of control group has been on decline.These results indicated that fatty acid metabolism may play a vital role in cold tolerance of large yellow croaker.Fatty acid composition was also detected and showed that the proportion of oleic acid(C18:1)of starvation group decreased slightly in the starvation stage,and increased significantly in the cooling stage,then decreased when maintained at 7°C.The oleic acid(C18:1)proportion of starvation group was significantly higher than the control group at the same phase(P<0.05).These results indicated that fatty acid metabolism might play a vital role in the response to cold stress.A full-length cDNA and genomic DNA of SCD(Stearoyl-CoA Desaturase)were cloned from large yellow croaker.The full-length cDNA was 1250 bp,including an open reading frame(ORF)of 1014 bp,encoded a polypeptide of 337 amino acid.The qPCR results showed,SCD gene was mainly expressed in liver,and then brain.The gene in liver increased significantly in the starvation stage,and the gene expression level of starvation group was significantly higher than control group.SCD gene expression in brain was not influenced by starvation stress,but the expression level of starvation and control group increased significantly in the cold stress stage,and the level of starvation group reached the maximum at 7 °C while control group at 9 °C.The western blot showed that SCD protein was expressed in liver and brain and it was consistent with the gene distribution.While SCD protein expression increased under starvation stress,it was inhibited at beginning of cold stress and then increased with the temperature decreasing.The expression level of control group declined significantly at 9 °C,and the level of starvation group remained higher level below 11 °C.Besides,the expression level of starvation group was higher than control group at 7 °C.In brain,the expression of SCD protein changed more significant,but the expression in brain was much lower than in liver.The results suggested that SCD functioned in liver,and affected the cold tolerance capacity.The subcellular location of SCD was detected in liver and brain,showed that SCD was localized in fat granules,mitochondria and granular endoplasmic reticulum of hepatic cells,while SCD in mitochondria of encephalic cells only.Three full-length cDNA and genomic sequences of GPDH gene were cloned by silico cloning and PCR.The qPCR results revealed that,cGPDH1 and mGPDH gene were expressed in most tissues of large yellow croaker and cGPDH2 gene was expressed mainly in muscle but not in liver.The cGPDH1 gene expression level in hepatic cell of starvation group was significantly lower than in control group,but there was no significant difference between two groups.Although the cGPDH2 and mGPDH gene expression in muscle up-regulated under cold stress,there was no significant difference between two group in the whole stage.The cGPDH1 and mGPDH gene expression level of starvation group changed more radically,the cGPDH2 gene expression of starvation group was significantly higher than control group.The ORF region and genomic sequence of CSDE1 gene was cloned by silico cloning,and verified by PCR.According qPCR,it showed that,CSDE1 gene was mainly expressed in muscle.The CSDE1 gene expression level of both groups increased under cold stress,but the starvation group was more quickly than control group in response to cold stress.The western blot showed the CSDE1 protein expression levels in brain,liver,heart and muscle of starvation group were significantly than control group.These results indicated that CSDE1 gene might be involved in cold tolerance mechanism of large yellow croaker,and starvation enhanced the gene expression.