| Abalone Haliotis discus hannai is an important economical specie. Along with the development, the abalone aquaculture suffered from serious problems such as frequent diseases and germplasm degradation. These problems restrict the further development of aquaculture industry. Studying the expressions of immune-related genes and understanding the immune defense mechanism can provide theoretical reference to improvement of anti-disease ability of H. discus hannai. As a common molecular biology method, florescent quantitative real-time PCR can detect the gene expression levels accurately, quickly and sensitively. The result can reflect the patterns of gene expression and help to better understanding their functions. However, the analysis of gene expression requires the calibration by stable reference genes. So the accurate result depends on the appropriate reference genes.In this study, 7 housekeeping genes including actin(ACT), 18 S ribosomal RNA(18S), cytochrome b(CYTB), elongation factor 1(EF1), glyceraldehyde 3-phosphate dehydrogenase(GAP), ribosomal protein S9(S9), and tubulin(TUB) were selected as candidates. The cycle threshold(Ct) values of these housekeeping genes in different organizations, growth stages and hemocytes post Micrococcus luteus and Listonella anguillarum stimulation were detected by florescent quantitative real-time PCR. The stabilities of these genes were analyzed by Bestkeeper, Normfinder and Genorm. The expression levels of C-type lectin from H. discus hannai in different tissues, growth stages and hemocytes posted bacteria stimulation were detected using selected genes as reference genes.Three softwares including Genorm, Normfinder and Bestkeeper were used to analyze the Ct values of housekeeping genes in gill, mantle, gonad, hemocytes, muscle and hepatopancreas of H. discus hannai. M values and stability values of these genes were calculated by Genorm and Normfinder, respectively. The stability ranking of these genes was ACT>CYTB>18S>TUB>EF1>GAP>S9. Standard deviations of the candidate genes were calculated by Bestkeeper. The result revealed that the stability ranking was CYTB>18S>TUB>ACT>EF1>GAP>S9. Considering the result of these softwares, CYTB was the suitable reference gene to detect the expressions of some gene in different tissues of H. discus hannai.The Ct values of seven Candidate genes from 2-cell, 4-cell, 8-cell, 16-cell, morula, gastrul, trochophore, veliger larvae, creeping larvae and juvenile abalone were obtained by florescent quantitative real-time PCR. Genorm and Normfinder were used to investigate stability of genes by analyzing Ct values. The result showed the ranking of the most stable to the least stable genes was ACT>CYTB>TUB >GAP>18S>EF1>S9. The stability ranking of candidate genes were analyzed by Bestkeeper. The ranking of the most stable to the least stable genes was TUB>ACT>GAP>18S>CYTB>S9>EF1. Stability ranking of three softwares was summed up. ACT was suitable gene as reference gene when the patterns of genes expression was detected in different growth stages.The Ct values of these genes in hemocytes posted bacteria stimulation were analyzed by Genorm, Normfinder and Bestkeeper. After M. luteus stimulation, the stability ranking of candidates were analyzed by Genorm and Normfinder. The result was ACT>CYTB>TUB>EF1>18S>GAP>S9. And the result of Bestkeeper was TUB>CYTB>ACT>EF1>18S>GAP>S9. After L. anguillarum stimulation, the stability ranking of candidate genes was analyzed by Genorm. The candidates were ranked as ACT>CYTB>TUB>EF1>18S>GAP>S9 according to their stability. While the stability ranking of candidate genes showed by Normfinder and Bestkeeper was ACT>CYTB>TUB>EF1>GAP>18S>S9 and ACT>TUB>CYTB>EF1>18S>S9>GAP, respectively. Taking results of three softwares together, ACT was suitable reference gene to measure the expressions of genes in hemocytes post bacteria stimulation.Using ACT and CYTB as reference genes, the relative expressions of C-type lectin in H. discus hannai from different tissues, different growth stages and hemocytes after M. luteus or L. anguillarum stimulation were measured. It revealed that the transcripts of C-type lectin were constitutively expressed in a wide range of tissues with different levels, with the highest levels in muscle and hemocytes. The expressions of C-type lectin were significantly upregulated in the period of morula and reached the peak in the period of creeping larvae. It might be related to the formation of hemocytes. M. luteus or L. anguillarum stimulation could induce up regulation in hemocytes of H. discus hannai. It implied that C-type lectin might participate in immune response against microorganisms.In summary, compared with other candidates, ACT and CYTB showed better stability and they can be used as reference genes to detect the expression patterns of some gene from H. discus hannai in different tissues, different growth stages and hemocytes after M. luteus or L. anguillarum stimulation. |
PDF Full Text Request