【摘要】 为在分子水平解析牙鲆(Paralichthys olivaceus)抗淋巴囊肿病的机理,本研究克隆牙鲆淋巴囊肿抗病免疫相关基因efhd2和tbc1d25的c DNA全序列,运用生物信息学方法对基因序列进行了详细分析;同时,采用荧光定量PCR分析了efhd2和tbc1d25基因在牙鲆胚胎发育不同阶段、淋巴囊肿抗病和患病个体不同组织的表达特征。结果显示,efhd2基因c DNA全长为5231 bp,开放阅读框(ORF)长为699 bp,编码232个氨基酸。tbc1d25基因c DNA全长为3173 bp,ORF长为2601 bp,编码866个氨基酸。定量结果显示,efhd2和tbc1d25基因在胚胎发育的各个时期均有不同程度的表达,其中,efhd2在出膜仔鱼期表达量最高,而tbc1d25在受精卵中的表达量显著高于其他时期(P<0.05)。在所研究的淋巴囊肿抗病和患病个体不同组织中,efhd2和tbc1d25基因均有不同程度的表达,抗病个体的血液中,这2个基因的表达量均显著高于患病个体(P<0.05)。本研究结果为深入探讨efhd2和tbc1d25基因功能和解析牙鲆淋巴囊肿抗病机理提供了基础资料。更多还原

【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 tbc1 d25. In this study, we cloned the full-length cDNA sequences of efhd2 and tbc1 d25 by using RACE(rapid-amplification of cDNA ends). The efhd2 gene was5231 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 tbc1 d25 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 TBC1 D25 have 71%, 74%, 72% and 74% homology with these four species, respectively. The expression profiles of efhd2 and tbc1 d25 were analyzed by quantitative real-time PCR(qRT-PCR). Both efhd2 and tbc1 d25 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 tbc1 d25 gene in the fertilized egg was significantly higher than that in other stages(P<0.05); during development, the expression level of tbc1 d25 decreased to the lowest level in late gastrula(P<0.05); there was no significant difference in the expression level of tbc1 d25 in late gastrula, sarcomere, and heartbeat stages(P<0.05). In the lymphocystis disease-resistant and-sensitive individuals, the expression of efhd2 and tbc1 d25 was detected in head, kidneys, liver, blood,gills, heart, gonads, muscle, intestine and spleen. The expression of efhd2 and tbc1 d25 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 tbc1 d25, as well as the mechanism of disease resistance of the Japanese flounder to lymphocystis disease.更多还原