大菱鲆家系选育二代7种免疫因子的分析

Analysis of seven immune-related genes in selective second filial families(F_2) of turbot(Scophthalmus maximus)

为选育高成活率的大菱鲆(Scophthalmus maximus)抗鳗弧菌(Vibrio anguillarum)群体,对30个大菱鲆选育二代家系(F2)和1个普通养殖群体进行鳗弧菌攻毒实验(周期为14 d),统计分析死亡率。选取死亡率不同的6个选育家系和普通养殖群体构成7个实验组,采用半定量-聚合酶链反应(semi-quantitative polymerase Chain reaction,RT-PCR)技术对它们肝脏、脾脏、头肾的相关免疫因子——溶菌酶(Lysozyme)、抗菌肽(Hepcidin)、热激蛋白70(HSP70)、热激蛋白90(HSP90)、免疫球蛋白(Ig M)、C-型凝集素(C-type lectin)、Lily-型凝集素(Lily-type lectin)的表达量开展研究,并应用SPSS 16.0软件对攻毒前后各免疫因子的表达量与攻毒后存活率之间的相关性进行分析。研究结果表明:攻毒前后选育家系幼鱼肝脏、脾脏、头肾中7个免疫因子的表达量普遍高于普通养殖幼鱼,且在7个实验组中,攻毒后的表达量相较攻毒前均呈现下降趋势;攻毒前,肝脏中lysozyme、Ig M的表达量与存活率为极显著性正相关(P<0.01),HSP70、HSP90、C-type lectin、Lily-type lectin的表达量与存活率为显著性正相关(P<0.05);脾脏中,Hepcidin、HSP70和HSP90的表达量与存活率为极显著性正相关(P<0.01);头肾中,HSP70的表达量与存活率为显著正相关(P<0.05)。综上,可以推断选育家系相对于普通养殖群体大菱鲆抗鳗弧菌性能更强,且7种免疫因子在鳗弧菌感染鱼体的过程中发挥重要的抗感染作用;从F2中获得一个抗鳗弧菌性能较强的家系(23号),可用于今后大菱鲆抗鳗弧菌品系的选育指导工作,为大菱鲆高成活率群体的选育提供参考资料和理论依据。

For breeding turbot group with high survival rates and resistance to Vibrio anguillarum, 30 selective second filial families（F2） with a high survival rate and one common culture group of turbot（Scophthalmus maximus L.） were infected with V. anguillarum. Six F2 and one control groups were picked out as samples for detecting the following immune responses. Gene expressions of seven immune-related factors（lysozyme, HSP90, HSP70, Ig M, hepcidin, C-type lectin, and lily-type lectin） were analyzed in turbot livers, spleens, and head kidneys using RT-PCR. Correlation analysis between the expression of these immune genes before infection and survival rate after challenge assays（period of 14 days） was carried out using SPSS 16.0 software. Results showed that regardless of samples infected with V. anguillarum, the expression of immune-related genes in the six F2 populations was generally stronger than that in the control and the expression status of most immune genes was higher in the seven experimental groups without Vibrio stress thanin the injected groups. The expression of the remaining six immune genes, except hepcidin, before Vibrio infection showed a significantly positive correlation with survival rate in livers（P 0.05） and the gene expression of lysozyme and Ig M exhibited a highly significant positive correlation（P 0.01）. Gene expressions of hepcidin, HSP70, and HSP90 showed a highly significant positive correlation with survival rate in spleens（P 0.01）; the expression of HSP70 exhibited a significant positive correlation with survival rate in head kidneys（P 0.05）. Therefore, F2 selective families with a high survival rate could possess an outstanding resistance against V. anguillarum and the seven immune-related genes could play important roles in bacterial resistance of turbot. We obtained a selective family（No. 23） with a strong resistance against V. anguillarum, which could guide selection of turbot strains with disease-resistance trait in the future and provide a reference and theoretical basis for turbot breeding.