Effects Of Salinity Change On Nutritional Components And Gene Expression Of Chinese Mitten Crab,Eriocheir Sinensis

Chinese mitten crab(Eriocheir sinensis)is an important economic aquatic species in China,The two important stages of Chinese mitten crab,breeding migration and growth migration,are both related to salinity changes.Salinity change would lead to osmotic pressure change,and osmotic pressure regulation has always been a hot research issue in the world.In this study,we used the nutrition,molecular biology,bioinformatics and other research methods to study expression stability of genes,nutritional components and transcriptome profiles of gill under different salinity condition in Chinese mitten crab.The aim is to investigate the molecular mechanism of osmotic pressure regulation and changes of nutrient composition in this species exposed in differenty salinity.The results are presented as follows: Part1 Study on expression stability of relative genes in Chinese mitten crab under different salinity stressIn order to study expression characteristics of osmoregulation related gene in Chinese mitten crab(Eriocheir sinensis)and to understand the molecular basis of osmotic regulation in migratory aquatic organisms.In this study,expression stability of ten relative genes were investigated in Chinese mitten crab under the four different salinity stress(0,10,20,30),including four energy metabolism genes;three growth related genes;two stress related genes;one osmotic regulating relative gene.The results showed that there were significant expression difference(P<0.05)under different salinity stress for the three energy metabolism related genes(VATB,UBE,GAPDH),the one stress related gene(GST)and osmoregulation gene(NAK),whereas no significant expression difference(P>0.05)were observed in the other reminding genes.Using the three software of BestKeeper,NormFinder and Genorm,the results indicated that these two genes(GST,NAK)were expression sensitive and instability under different salinity stress,but the three growth related genes(S27??-ACTIN??-TUB)were high expression stability under different salinity stress,implying this two genes could be used as reference genes in environment stress study(e.g salinity stress)for Chinese mitten crab.The present study provided useful information for observing expression stability of different categories of genes and screening reference genes in Chinese mitten crab under salinity and other environmental stress.Part2 Effects of salinity on nutritional components of Chinese mitten crabIn order to study the effect of salinity on the nutritional components of Chinese mitten crab(Eriocheir sinensis),this chapter compared the composition of fatty acids and amino acids in muscle and hepatopancreas of Chinese mitten crab under different salinity levels(0,20).The results showed that the content of monounsaturated fatty acid(MUFA)and polyunsaturated fatty acid(PUFA)under salinity 0 were significantly higher(P<0.01)than those under salinity 20 in muscle,indicating that salinity stimulation reduced the content of unsaturated fatty acids in muscle.However,there were no significant differences(P> 0.05)of the content of fatty acids in the hepatopancreas under different salinity levels,indicating salinity stimulation had little effect on the amount of fatty acid in hepatopancreas.The content of essential amino acids(EAA)under salinity 20 was significantly higher(P< 0.05)than that under salinity 0.Salinity stimulation had little effect on the content of amino acids in hepatopancreas of female crabs,but in male crab,the contents of essential amino acids and non-essential amino acids under salinity 20 were strikingly higher(P< 0.05)than those under salinity 0,indicating that salinity stimulation had more effect on amino acids of male crabs.The effects of salinity stimulation on the content of amino acids and fatty acids in hepatopancreas and muscle tissue illustrated that salinity could plays a important role in nutritional quality of Chinese mitten crab.Part3 Transcriptome analysis of Chinese mitten crab at different salinity levelsChinese mitten crab is a kind of migratory economic crab with migrating twice during lifetime which are referred to as growth migration(anadromous migration,nearly salinity 0)and breeding migration(catadromous migration,nearly salinity 20).In this study,the two different migration process were simulated based on the three salinity conditions(0,20,0),then Illumina Hiseq 4000 technology was used to sequence under the three salinity conditions mentioned above and the data was analyzed to obtain the transcriptome data of high quality.As a result,the three sequences sized at 92,612,226 bp and around 40 Gb sequence data were acquired.These sequences were then assembled by De novo and 70,214 transcripts were gained with 42,188 transcripts annotated in the database of NCBI-NR.The total 783 differentially expressed genes were identified under different salinity conditions by analyzing of genes differential expression,in which 91 genes were selected as highly expressed genes under salinity 0 and 93 genes were identified as highly expressed genes under salinity 20.Moreover,62 highly expressed genes were found under the salinity 0-20-0.The differentially expressed genes,sodium and potassium ATP enzyme beta subunit are involved in regulating the osmotic pressure,maintaining the internal environment of cells;The heat shock protein 67b2 gene could adapt salinity changes,and the NADH dehydrogenase gene provides energy for the regulation of osmotic pressure.Generally,the transcriptome data of Chinese mitten crab under different salinity conditions provided a reference for understanding the molecular mechanism of osmotic pressure adjustment in Chinese mitten crab.