Abstract:The Japanese flounder (Paralichthys olivaceus) is one of the most important marine culture species in China. However, the outbreak of diseases has seriously affected the industrial culture of this particular fish. Among these diseases, the one caused by the lymphocystis disease virus has been spreading widely and has resulted in severe economic losses every year. In order to select new varieties of Japanese flounder that are resistant to lymphocystis disease in China, and to elucidate the mechanism of disease resistance at the molecular level, we used a high-throughput sequencing technique to analyze the transcriptome of kidney tissues of the Japanese flounder, and screened out a number of functional genes closely related to resistance, including efhd2 and tbc1d25. In this study, we cloned the full-length cDNA sequences of efhd2 and tbc1d25 by using RACE (rapid-amplification of cDNA ends). The efhd2 gene was 5231 bp in length, of which the length of the 5' untranslated region (5' UTR) was 142 bp and the length of the 3' UTR was 4390 bp. The open reading frame (ORF) was 699 bp in length, and encoded 232 amino acids with a molecular weight of 26.4 kDa and an isoelectric point of about 5.08. The full length of the tbc1d25 gene was 3173 bp, of which the 5' UTR length was 108 bp, and the 3' UTR length was 464 bp. The ORF was 2601 bp in length and encoded 866 amino acids with a molecular weight of 96.4 kDa and an isoelectric point of about 5.47. Multiple sequence alignment revealed that the amino acid sequences of EFHD2 have 83%, 88%, 73%, and 72% homology with Danio rerio, Oryzias latipes, Mus musculus, and Homo sapiens respectively; and those of TBC1D25 have 71%, 74%, 72% and 74% homology with these four species, respectively. The expression profiles of efhd2 and tbc1d25 were analyzed by quantitative real-time PCR (qRT-PCR). Both efhd2 and tbc1d25 were expressed at the fertilized egg, 4-cell, 32-cell, 128-cell, high blastocyst, low blastocyst, early gastrula, late gastrula, sarcomere, heartbeat, and hatched larva stages. The expression level of efhd2 in the 4-cell, low blastocyst and early gastrula stage was lower than that in other stages (P<0.05); the expression level of efhd2 began to increase from the sarcomere stage and reached the highest level at the hatched larva stage, and was significantly higher than that in other groups (P<0.05). The expression level of the tbc1d25 gene in the fertilized egg was significantly higher than that in other stages (P<0.05); during development, the expression level of tbc1d25 decreased to the lowest level in late gastrula (P<0.05); there was no significant difference in the expression level of tbc1d25 in late gastrula, sarcomere, and heartbeat stages (P<0.05). In the lymphocystis disease-resistant and -sensitive individuals, the expression of efhd2 and tbc1d25 was detected in head, kidneys, liver, blood, gills, heart, gonads, muscle, intestine and spleen. The expression of efhd2 and tbc1d25 was significantly higher in the blood of lymphocystis disease-resistant individuals than in the lymphocystis disease-sensitive individuals. This study provides a basis for further studying the gene function of efhd2 and tbc1d25, as well as the mechanism of disease resistance of the Japanese flounder to lymphocystis disease.
