| In this paper,the changes in fatty acid content of the adductor muscle of Pinctada fucata martensii before and after nuclear implantation were detected and analyzed.Combined with the data of blood cell transcriptome before and after nuclear implantation,the effect of polyunsaturated fatty acids on immunity of P.f.martensii was discussed from the point of view of lipid metabolism.Then the genes related to polyunsaturated fatty acid synthesis of P.f.martensii were cloned and their functions were analyzed.The main results are as follows:(1)A total of 18 free fatty acids were detected in the adductor muscle of P.f.martensii before and after nuclear implantation.The contents of ?-linolenic acid,arachidic acid,linoleic acid,vaccenic acid,oleic acid,stearic acid,heptadecanoic acid,palmitoleic acid,palmitic acid,pentadecanoic acid,myristic acid,lauric acid,eicosadienoic acid,docosadienoic acid,and 11,14,17-eicosatrienoic acid were not significantly different before and after nuclear implantation.The contents of arachidonic acid,eicosapentaenoic acid and docosahexaenoic acid increased significantly after nuclear implantation(P < 0.05).193 genes related to lipid metabolism were found by blood cell transcriptome analysis of P.f.martensii before and after nuclear implantation,such as the elongation of very long-chain FA protein 5,acyl-Co A 6-desaturase,glycerol-3-phosphate O-acyltransferase,phospholipase A2,cytochrome P450 and prostaglandin-H2 D-isomerase.Pathway enrichment analysis showed that these genes were mainly involved in "biosynthesis of unsaturated FAs"," FA biosynthesis ","ARA metabolism" and " glycerolipid metabolism".These results suggest that P.f.martensii may need more polyunsaturated fatty acids to regulate its inflammation and immune response after nuclear implantation.Therefore,feeding P.f.martensii with polyunsaturated fatty acids or selecting P.f.martensii with strong biosynthesis of unsaturated fatty acids for bead breeding may be helpful to the recovery after nuclear implantation.(2)In view of the important role of polyunsaturated fatty acids in the nuclear immunity of P.f.martensii,the genes related to the synthesis of polyunsaturated fatty acids,Pm-5FAD,Pm-FAD2,Pm-DES1 and Pm-DES2,were cloned and analyzed in this paper.The results showed that these genes could respond to the nuclear stress response of P.f.martensii.The full length of Pm-?5FAD sequence is 2 350 bp,which encodes 433 amino acids and has typical Cyt-b5 and FA?desaturase domains.The amino acid sequence of ?5FAD among species has high homology,including one heme binding motif and three histidine family conserved motifs,and highly expressed in gonad,mantle central region and gill.The expression level increased significantly at 1 day after nuclear implantation(P < 0.05),decreased significantly at 3 days after nuclear implantation compared with 1 day after nuclear implantation,and tended to be stable after 15 days.Yeast eukaryotic expression vector was constructed by Gateway technology,and Pm-?5FAD was successfully introduced into Saccharomyces cerevisiae host cells.The results of functional verification show that Pm-?5FAD has catalytic activity of ?5Fad,which can convert C20:3n-6 substrate to C20:4n-6,and the substrate conversion efficiency is about 20.78%.The full length of Pm-FAD2 sequence is 1 731 bp,which encodes 435 amino acids,has typical Cyt-b5 and FA?desaturase domains,FAD2 has high homology among species,including one heme binding motif and three histidine conserved motifs,the primary and advanced structure of Pm-FAD2 is conserved,and it is highly expressed in gonad,gill and mantle central region.The expression level increased significantly at 1 day after nuclear implantation(P < 0.05),then decreased gradually,and tended to be stable after 15 days.Yeast eukaryotic expression vector was constructed by Gateway technology,and Pm-FAD2 was successfully introduced into Saccharomyces cerevisiae host cells.The full-length Pm-DES1 sequence is 1 141 bp,which encodes 340 amino acids;Pm-DES1 contains Lipid?DES and FA?desaturase conserved domains;the highest homology between Pm-DES1 and Crassostrea virginica is 79.07%,and each species contains three conserved histidine families;its expression increased significantly 3 days after nucleation(P < 0.05),then decreased gradually,and decreased to the pre-nuclear expression level on the 30 th day.The full length of Pm-DES2 sequence is 1501 bp,which encodes 331 amino acids,contains Lipid?DES and FA?desaturase domains,contains three conservative histidine families,and the sequences of histidine groups have high homology among species.Pm-DES2 is highly similar to Pomacea canaliculata in space.The expression was significantly high in gonad and Gill(P < 0.05),and had obvious tissue specificity.The expression increased significantly at 3 days after nuclear implantation(P < 0.05),then decreased gradually,and tended to be stable after 15 days. |
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