Molecular Cloning And Characterization Of Dicer-1 From White Shrimp Litopenaeus Vannamei

Viruses are the most common pathogens of penaeid shrimp, and viral diseases have caused the substantial economic losses. RNAi-mediated silencing has been proven to be a highly conserved defence mechanism, that results in the suppression of gene expression through the sequence-specific degradation and repression of the homogenous of virus mRNA. Dicer is a member of RNAase III family and the key initiative enzyme in RNAi pathway. Dicer can cleave dsRNA into small RNAs categorized as small interfering RNAs (siRNAs) or micro interfering RNAs (miRNAs), which can guide specific RNA silencing. The main objectives of this study were to clone the full length cDNA of Dicer-1 gene (designated as LvDcrl) from white shrimp Litopenaeus vannamei, to investigate the mRNA expression pattern of LvDcrl under Taura syndrom virus (TSV) infection and the profile of Dicer-1 expressions in larval development. The LvDcrl mRNA expression pattern would be helpful for the further research on the relation of host and virus and the ontogeny of immunity, as well as designing efficient strategies for virus disease control.The full-length cDNA of from LvDcrl was of 7636 bp nucleotides with a poly A tail, a 5' UTR of 136 bp, a 3'UTR of 78 bp, and ORF of 7319 encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all the conserved functional domains including a helicase, a domain of unknown function (DUF283), a PAZ (Pinwheel-Argonaut-Zwille) domain and RNase domain. LvDcrl shared 97.7% similarity with Pm Dcrl from Penaeus monodon. Quantitative RT-PCR analysis showed that LvDcrl mRNA was expressed higher in hemolymph than that in hepatopancreas, muscle, intestine, brain, gill of adult shrimps. The mRNA expression of LvDcrl during larval development, ranging from fertilized eggs to post-larvae, was also recorded. In early larval stages, the expressions level of LvDcrl fluctuated regularly following Naupliusâ…¤>Naupliusâ… , Zoeaâ…¢>Zoeaâ… , Mysisâ…¢>Mysisâ… (P>0.05). In whole larval stage, the expressions LvDcrl showed two obvious turning points. The first one was from Naupliusâ…¤to Zoeaâ… , a key transition from endogenesis to exogenesis nutritional stage, and LvDcrl expression sharply clined down. The second one was observed from mysis to post-larval stages, and LvDcrl expression in post-larval stage increased to high level. The expression of LvDcrl showed specific-stage difference during larval developmental stages. After TSV challenge, the mRNA expression of LvDcrl in hemocyte was significantly (P<0.05) increased comparing to the control group at 4 h. The highest mRNA level was observed at 8 h which was 8.77-fold higher than that in the control shrimps (P<0.05). The mRNA expression level of LvDcrl in the gills increased at 12 h and reached maximum at 24 h which was significantly 6.74-fold higher than the control, and then decreased and recovered to the original level at 36 h. The results indicated that LvDcrl was a constitutive and inducible expressed protein that could be involved in shrimp immune response against virus infection.The splicing virants of LvDcrl mRNA were indentified in shrimps infected by TSV. The retion of intron lied in RNase III catalytic sequences, and the mis-splicing of exon in helicase catalytic sequences of mRNA resulted in two transcripts with different length. The un-cleaving and mis-splicing transcripts could not execute the activities of RNase and helicase perfectly to cleave virus dsRNA, and the anti-virus efficency of RNAi was impaired. It was suspected that there was some mechanism for the virus to escape from LvDcrl and the related RNAi-pathway.