| Iron is a vital nutritional element,essential for a great variety of important biological processes.Conversely,iron's redox properties play a role in its toxicity.Through participation in Fenton chemistry,iron produces reactive oxygen species,which leads to DNA strand breakage,lipid peroxidation,and protein denaturation.This study aims to investigate the biological processes affected by a prolonged iron diet and identify differentially expressed genes in the silkworm after feeding with iron-loaded diet(10 and 100 ppm Fe)by microarray analysis.By correlating the altered expressions of genetic variants of the whole-transcriptome profile after feeding on iron and normal silkworm diet,both up-regulated and down-regulated genes were identified.The objective of this project was not only to identify these novel genes but to observe the external biological changes during iron supplementation period of growth of the silkworm.The newly hatched silkworms were fed on to cocooning.Diet containing Fe was prepared by spraying Ferric chloride(FeCl3)aqueous solution on fresh mulberry.On the fourth day of the fifth instar,the silkworms were weighed.The result showed that the weight of 1,10 and 100 ppm Fe group was greater 14.7%(P <0.01),20.0(P <0.01)and 18.14%(P <0.01)than the control group in the fourth day of the fifth instar,respectively.While on the contrary,1000 ppm group not only grew slowly but also died due to high toxic metabolism caused by iron overload.ICP-AES was used to detect the hemolymph iron levels in the 1,10,100 ppm Fe group which corresponded to 119.7%,165.3%(P <0.05)and 213.5%(P <0.01)compared to those of the control group,respectively.Antibody against ferritin H subunit was used to perform Western blot analysis of hemolymph protein.The results showed that a specific band was observed clearly at 1,10,100 ppm group,while the control group showed no band,which indicated that the model of iron overload in silkworm was effective and usable.Further whole-transcriptome analysis was done to give better insights on biological pathways and molecular mechanisms of iron at gene level in insects.Microarray analysis was employed to extensively identify the 523 and 326 differentially expressed genes that were observed in 10 and 100 ppm Fe group compared to the control,respectively.Amongst these genes,249 are common between the 10 and 100 Fe group,also 152 and 97 of these genes are up-regulated and downregulated,respectively.The GO analysis indicated that 79 genes of both the upregulated genes,49 genes of both the down-regulated genes were found with at least one common gene,respectively.52 genes of both the up-regulated genes and 8 genes of both the down-regulated genes were involved in 78 and 14 KEGG pathways,respectively.The common genes involved were 12 serine proteases and serine proteases homologs,19 known Fe regulated,24 immune-related,41 cuticular and cuticle genes.After the whole transcriptomic analysis a varying number of genes(carboxypeptidases A,fibrohexamerin/P25,serine protease homologs 85,fungal protease inhibitor F,transferrin,sex-specific storage-protein 2,cuticle protein CPH45,insect intestinal mucin,peptidoglycan recognition protein B and the unknown gene)were discovered to be involved in the regulation of iron overload responses that help to avoid imbalances in ion distribution.The gene expression levels of the 10 genes were validated by designing specific primers for RT-qPCR in the fourth day of the fifth instar of control,10,100 ppm group.The results indicated that the change tendency of the expression levels of the 10 genes has similar patterns with the changes measured by the microarray. |
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