Cloning,Expression Analysis And SNPs Detection Of Low Salinity-tolerant Genes In Black Tiger Shrimp(Oenaeus Monodon)

To study the molecular mechanism of low salinity toleration and to screen molecular marker for new variety breeding in Penaeus monodon,three genes related to low salinity toleration were first cloned and analyzed by bioinformatics.Then,the expression of three genes in different tissues and gill during low salinity stress were detected by fluorescence-quantitative real time PCR.According to the P.monodon cDNA Library constructed by my laboratory,the full length cDNA of the three genes was obtained by RACE(rapid amplification of cDNA ends)technique.Then,use the genomicDNA to design primers to amplify the genomic sequence and use wild population samples to screen SNPs on the genomic sequences of the three genes.Good quality sites were selected for SNP genotyping and association analysis with low salinity tolerance.The cDNA of P.monodon glucose transporter-1(PmGLUT1)was 2023bp,a 276bp of 5’non-coding region(UTR),a 271bp of 3’UTR,a poly(A)tail with 23bp.The length of the open reading frame(ORF)was 1476bp encoding a polypeptide of 491 amino acids.The molecular mass of the deduced amino acid(aa)sequence was 54.179kDa with an estimated theoretical isoelectric point of 6.39.There were 31 phosphorylation sites and 1glycosylation site with 12 predicted transmembrane helical structures in this protein.Multiple sequence alignment and phylogenetic analysis showed that the PmGLUT1 with the GLUT1 of Litopenaeus vannamei shared a similarity of 99.59%.From zygote to postlarva stages,the expression of PmGLUT1 fluctuated but showed an increasing trend.The analysis of ecdysis showed that the expression level of PmGLUT1 mRNA in gill was the highest in the intermolt stage,followed by the postmolot stage,and the lowest in the molting stage.The PmGLUT1 was expressed in all tested tissues with the highest expression in gill tissue,followed by hepatopancreas,and the lowest expression in ovary.After 96h acute low salinity stress,the expression level of PmGLUT1 mRNA in hepatopancreas was significantly higher than that in the control group,while the expression level in gill was significantly lower than that in the control group(P<0.05).The cDNA of P.monodon glucose transporter-2(PmGLUT2)was 2018bp,a 94bp of 5’UTR,a 352bp of 3’UTR,a poly(A)tail with 29bp.The length of the open reading frame(ORF)was 1572bp encoding a polypeptide of 523 amino acids.The molecular mass of the deduced amino acid(aa)sequence was 56.568kDa with an estimated theoretical isoelectric point of 4.96.There were 55 phosphorylation sites and 4 glycosylation sites with 12 predicted transmembrane helical structures in this protein.Multiple sequence alignment and phylogenetic analysis showed that the PmGLUT2 with the facilitated trehalose transporter Tret1-like of L.vannamei shared a similarity of 94.77%,while the PmGLUT2 with the GLUT2 of L.vannamei shared a similarity of 60.54%.From zygote to postlarva stages,the expression of PmGLUT2 decreased first and then increased,but there was no significance.The analysis of ecdysis showed that the expression level of PmGLUT2 mRNA in gill was the highest in the intermolt stage,followed by the molting stage,and the lowest in the premolt stage.The PmGLUT2 was expressed in all tested tissues with the highest expression in lymph tissue,followed by gill,and the lowest expression in ovary.After 96h acute low salinity stress,the expression level of PmGLUT2 mRNA in hepatopancreas and gill was inhibited.The cDNA of P.monodon Na~+/K~+/2Cl~-cotransporter(PmNKCC)was 3613bp,a 48bp of UTR,a 382bp of 3’UTR,a poly(A)tail with 30bp.The length of the open reading frame(ORF)was 3183bp encoding a polypeptide of 1060 amino acids.The molecular mass of the deduced amino acid(aa)sequence was 116.805kDa with an estimated theoretical isoelectric point of 6.29.There were 101 phosphorylation sites and 11 glycosylation site with 11 predicted transmembrane helical structures in this protein.Multiple sequence alignment and phylogenetic analysis showed that the PmNKCC with the NKCC of Marsupenaeus japonicus shared a similarity of 93.68%.From zygote to postlarva stages,the expression of PmNKCC fluctuated periodically with the development of larval developmental morphology.The analysis of ecdysis showed that the expression level of PmNKCC mRNA in gill was the highest in the postmolot stage,followed by the premolt stage,and the lowest in the molting stage.The PmNKCC was expressed in all tested tissues with the highest expression in gill tissue,followed by lymph,and the lowest expression in muscle.After 96h acute low salinity stress,the expression level of PmNKCC mRNA in hepatopancreas,gill,gut and stomach tissues increased first and then decreased with the change of low salinity stress time.RNA interfere analysis showed that injection of ds PmNKCC reduced the expression of PmNKCC and increased mortality of P.monodon during acute low salinity stress.Based on the obtained cDNA full lengths of PmGLUT1,PmGLUT2 and PmNKCC,the genomic sequences of the three genes were amplified using genomicDNA as a template.In this research,partial genomic sequences of three genes were amplified for the first time.The partial genomic sequence of PmGLUT1,PmGLUT2 and PmNKCC was2148bp,2837bp and 4888bp respectively.There were 3 single nucleotide polymorphism(SNP)loci detected by direct sequencing method in PmGLUT1 genomic sequence;There were 28 SNP loci detected by direct sequencing method in PmGLUT2genomic sequence;There were 66 SNP loci detected by direct sequencing method in PmNKCC genomic sequence.Good quality SNPs were selected for SNP genotyping and association analysis.PmNKCC 2061 and PmNKCC 2157 of PmNKCC were significantly different in the capacity of acute low salinity toleration(P<0.05).The results of our studies have indicated that PmGLUT1,PmGLUT2 and PmNKCC might play an important role in low salinity toleration metabolism and be involved in responses to acute low salinity stress,PmNKCC 2061 and PmNKCC 2157 can be used as salinity toleration candidate SNP loci for further testify in next generation of P.monodon.And we expect this two SNPs can be used in the future Penaeus monodon breeding.