| The silkworm has a long history of research and a clear genetic background.It is the third lepidopteran model insect and an important economic insect to complete the whole genome sequencing after Drosophila and Anopheles.Silkworm,as an oligotrophic insect,almost has good feeding properties on its natural mulberry leaves.However,the feeding habits of silkworms on non-host plants and artificial feed vary greatly between varieties and even individuals,and are also affected by many factors.The feeding of silkworm is a complex physiological process.In addition to food factors,the regulation of the expression of metabolic related genes is an intrinsically important factor.Up to now,The silkworm artificial feed metabolism related gene has not been obtained,and its molecular mechanism has not been fully clarified.In this research,the early screening of the laboratory was used to study the high-feeding silkworm variety GS that does not contain mulberry leaf powder artificial feed.Screening silkworm metabolism related genes,there are mainly the following 5 points:1)According to the analysis of metabolome data,the silkworm silkworm GS breeds fed by artificial feed lack essential amino acids,amino acid metabolites are enriched,vitamins are deficient,glucose metabolism is disordered and metabolites are enriched,and lipid metabolism is reduced.It is the silkworm GS variety and the silkworm C1015 variety fed by mulberry leaves.The content of essential amino acids in the silkworm GS variety is higher than that of the silkworm C1015 variety.It shows that the silkworm GS variety has a greater protein metabolism capacity than the silkworm C1015 variety,making it suitable for the ratio of more protein and less sugar in artificial feed.2)A total of 331 differential genes were screened at the m RNA level in the silkworm GS after mulberry leaves and artificial feed breeding,including 102 up-regulated genes and 229 down-regulated genes.Among the 331 differential genes,the most enriched and annotated GO is the metabolic process in biological processes,while KEGG is the most annotated cytochrome P450 metabolic pathway for xenobiotics,the conversion pathway between pentose and glucuronic acid,glycerides Metabolic pathways and protein processing pathways in the endoplasmic reticulum.T here are 1311 differential genes in C1015 and GS which are also mulberry leaves,including 716 up-regulated genes and 595 down-regulated genes.The 1311 differential gene GO enriched annotations are the metabolic processes in biological processes and the catalytic activity of molecular functions.The KEGG enriched annotations are the lysosomal pathway,the protein processing pathway in the endoplasmic reticulum,the glutathione metabolism pathway,the starch and sucrose metabolism pathway,and the pentose and glucuronic acid mutual conversion pathway.Statistics on the SNP mutation sites of several samples found that all the SNP mutation sites mainly occurred in the genic region,and occurred in the downstream in the manner of synonymous mutation.62% of the mutations are conversions,and 38% are transversions.There are 12 single nucleotide variations,mainly A / G,G / A,C / T,and T / C,which account for 65.71% of the total.C / G and G / C have the least variation,accounting for 8.20% of the total.Other mutation types accounted for 26.09% of the 12 single nucleotide mutations.3)The results of the joint analysis of omics showed that six differential genes were found in the arginine and proline metabolism pathway maps,namely phospholipid transport ATPase,spermidine synthetase,pyrroline-5-carboxylic acid reductase,S-Pro-adenosylmethionine decarboxylase,prolyl 4-hydroxylase alpha subunit,Silkwormnew Gene406.4)The three genes of spermidine synthetase,pyrroline-5-carboxylic acid reductase and prolyl 4-hydroxylase alpha subunit were cloned and the expression analysis of each tissue.The amino acid comparison showed that,compared with the silkworm C1015,the amino acid sequences of the three genes cloned in the silkworm GS were all difference.Tissue expression analysis showed that the expression of the pyrroline-5-carboxylic acid reductase gene of the silkworm in the two tissues was not significantly different;the proyl 4-hydroxylase alpha subunit of the silkworm was in the two breeds.There were significant differences in the expression in the three tissues of the genital duct and gonad;there was a significant difference in the expression of the midgut and fat body of the two varieties of silkworm spermidine synthase.Bioinformatics analysis showed that compared to the silkworm C1015,the prolyl 4-hydroxylase alpha subunit and spermidine synthase secondary structure of the silkworm GS variety lacked part of the ?-sheet,protein molecular mass,etc There are differences between the electric point and the phosphorylation site.5)In order to further analyze the function of spermidine synthetase,RNA interference of spermidine synthase was performed in GS varieties.Compared with the control,after feeding with artificial feed,GS interfered with the individual weight loss of 11%,cocoon production decreased by 36.8%,and mortality increased by 66.7%.Real-time quantitative PCR detection found that in interfering individuals,the m RNA levels of pyrroline-5-carboxylic acid reductase(FC=0.45)and S-adenosylmethionine decarboxylase(FC = 0.43)related to spermidine synthase were increased.The relative expression level has been reduced.SNP mutations in spermidine synthase(isoleucine to valine(170aa)and aspartate to threonine(241aa))not only change the secondary structure of spermidine synthase,but also It affects the activity of its enzymes,and also affects the expression and enzyme activity of its upstream and downstream genes,and jointly regulates the amino acid metabolism of silkworm,so that the silkworm GS variety strengthens the amino acid metabolism ability,so that it can adapt to the high protein and low sugar content in artificial feed.Proportion,showing a certain degree of extensive eating. |
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