| N-3 polyunsaturated fatty acids (n-3 PUFAs) are closely related to human health, but n-3 PUFAs biosynthetic pathways do not exist in humans and mammals. Therefore, n-3 PUFA must be obtained from the diet. In 1997, the gene of encoding n-3 polyunsaturated fatty acid desaturase was found by Spychalla et al. from elegans, and named as fat-1. Subsequently, many fat-1 transgenic animals rich with n-3 PUFAs were successfully generated by transgenic technology, which showed the feasibility for supplying the n-3 PUFAs to human through the transgenic animal food. With the large number of birth for various fat-1 transgenic domestic animals, attention has been paid to the biosafety of using these transgenic domestic animals.The self-health of transgenic domestic animals is an important evaluation content of biological safety. So far, although some studies had been reported on the evaluation of self-health and safety in transgenic domestic animals, these studies only limited to certain aspects of health state.Therefore, the current studies still have their own limitation for evaluating the self-health in transgenic domestic animals systematically and completely. In the present study, three living fat-1 transgenic cow were generated by transgenic somatic cell nuclear transfer (SCNT) at last.Integration sites analysis of foreign gene was performed for one transgenic cow by using high throughput sequencing technology, which found that the fat-1 gene was integrated into 15726078 bp for chromosome 16 of cow genome. Furthermore, the systematic study was performed for the three fat-1 transgenic cow at different angles, including the affected blood biochemical parameters, the detection of foreign gene drift, the affected levels of gene expression and plasma proteins in blood, as well as the gut microflora. Results showed that the regulation role of fat-1 gene for lipid metabolism, immune, cardiovascular system and anti-stress were presented in the three transgenic cow. The results built a practical safety evaluation system of self-health on large transgenic livestock, and provided a valuable reference for obtaining healthier and safer transgenic livestock.1. Generation and routine analysis of fat-1transgenic cowNine calves were generated by transgenic somatic cell nuclear transfer,6 were identified as fat-1 transgenic cow. The elevated n-3 PUFA level and the reduced ratio of n-6/n-3 PUFA were detected in the fat-1 transgenic cow through fatty acid analysis. Results of blood biochemical levelsindicated that the ALT was significantly lower in 6-monthes old transgenic cow than in wild-type cow and the AST, GLU, TC and LDL-L were significantly reduced in 18-monthes old transgenic cow compared with wild-type cow.The results also showed that the fat-1 gene was not detected by polymerase chain reaction (PCR) in the microbial DNA extracts of gut faeces, the transgenic cowshed topsoil and the topsoil of different locations at a distance 200 m from the transgenic cowshed.2. Integration sites analysis of fat-1 transgenic cowIntegration sites analysis of foreign gene was performed in FD006 transgenic cow by using high throughput sequencing technology. The results showed the single copy fat-1 gene was integrated into 15726078 bp for chromosome 16 of cow genome. Based on the analysis results for the sequence reads of integration sites and nearby it, the FD006 transgenic cow was identified as heterozygote. Results of the PCR also confirmed that the fat-1 gene was integrated into 15726078 bp for chromosome 16 of cow genome, and was a heterozygous transgenic cow. The study provided a technical route and theoretical basis for the integration sites analysis, target integration and stable expression of foreign gene in transgenic cow.3. Study for the affected genes in fat-1 transgenic cowTotal RNA was extracted from the blood of fat-1 transgenic cow and wild-type cow for analysis the affected genes by using the expression profile microarray.Results indicated that 2042 genes were identified as being significantly differentially expressed in fat-1 transgenic cows. Of the 2042 genes, there were 797 genes that were significantly up-regulated in the fat-1 transgenic cow than in the wild-type cow, while other 1245 genes were down-regulated. To further insight into the potential influence of the 1245 genes with significant difference in expressions, GO enrichment analysis was performed. Totally,90 GO Terms were significantly enriched, which were mainly related to host’s lipid metabolism, cell behavior, and immune and nervous systems development. Thirty-six key genes related to lipid metabolism were detected in 8 GO Terms. Furthermore, the’PPAR signaling pathway’ was obtained by the KEGG analysis, which was closely related to lipid metabolism of unsaturated fatty acid. The reliability of the microarray data was confirmed by the Real-time PCR for 16 genes related to lipid metabolism.Together, the results showed that the changes of gene expressions occurred after the fat-1 gene delivered into transgenic cow. These expression changed genes were related to these regulation roles in lipid metabolism, immune, and nervous systems development.4. Study of plasma proteomics in fat-1 transgenic cowDifferentially expressed plasma proteins of fat-1 transgenic cow were analyzed by the two-dimensional gel electrophoresis (2-DE) and mass spectrometry technique. Results showed that 15 differentiallyexpressed proteins were identified. GO and KEGG enrichment analysis were performed using the 15 differentially expressed proteins and their interaction proteins.Results showed that these proteins played important roles in lipid metabolism, immune, stress, nervous systems development and blood coagulation. Among 18 biological pathways related to lipid metabolism,12 biological pathways enriched the APOA1 protein, indicating the possible correlation between fat-1 and APOA1 for the regulation of lipid metabolism. Accordingly, the significantly higher plasma level of APOA1 was detected in fat-1 transgenic cow than wild-type cow.Then, the plasma level of APOA1 was negatively related to the plasma level of low density lipoproten-cholesterol/Total cholesterol (LDL-C, r=-0.90), whereas it was positively related to the ratio of high density lipoproten-cholesterol/Total cholesterol (HDL-C/TC, r=0.69). These results showed the fat-1 gene changed the expression for some plasma proteins in transgenic cow. In addition, the results aslo showed a potential regulation mechanism of the fat-1 gene for the lipid metabolism through mediating the expression of APOA1 in transgenic cow.5. Study of gut microbial community in fat-1 transgenic cowThe gut microbiota composition and diversity were compared between 3 fat-1 transgenic cow and 3 wild-type cow by sequencing the V4 variable region of 16s rDNA. Results indicated 9714 OTUs were obtained in both fat-1 transgenic cow and wild-type cow. The OTUs (8907) were detected lower in fat-1 transgenic cow than in wild-type cow (9488).Detection on species diversity indexes of gut microbes indicated that Chao and Shannon were significantly lower in fat-1 transgenic cow than in wild-type cow (P<0.05). Further analysis found that the changes of the gut microbiota composition and abundance were also detected in fat-1 transgenic cow. Results of the species abundance indicated that 3 phylum (Proteobacteria, Spirochaetes and Euryarchaeota) and 9 genera (Odoribacter, Methanobrevibacter, Lutispora, Spirochaetes, Bacteroides, Treponema, Dorea, Roseburiaand Acetitomaculum) were significantly differenced (p<0.05). Good correlations were detected in Dorea, Roseburia, Succinivibrio and Alistipes with the changed levels of plasma GLU and plasma lipids. In addition, the Odoribacter related to stress was significantly reduced in fat-1 transgenic cow. Take together, the expression of fat-1 gene changed the diversity, composition and abundance of gut microflora in transgenic cow. The changes of gut microflora suggested the emerging roles in metabolisms of glucose and lipids, as well as anti-stress, which also presented a possible regulation mechanism of the fat-1 gene for the lipid metabolism through changing the composition of gut microbiota in transgenic cow. |
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