Proteomic Analysis Of Midgut Reared With Artificial Diet And Mulberry Leaf,and Function Of Three UGT Genes In Bombyx Mori

At present,the production performance and resistance of silkworm rearing with full-age artificial diet are still obviously inferior to those of mulberry leaf rearing,and factory rearing of silkworm rearing with full-age artificial diet is the future development direction of sericulture.It is the main research objective to make silkworm rearing performance of full-age artificial diet reach or close to full-age mulberry leaf rearing level.The difference of nutrients in artificial diet and mulberry leaves may be the key factor affecting the growth and cocoon quality of silkworm.Therefore,in this study,i TRAQ technology was used to perform proteomics research on the midgut of silkworm fed with artificial diet and mulberry leaf,and the significant indigenous differential proteins were screened.Molecular docking was used to select the anti-nutritional factors that stably bind to UGT40B4,UGT340C2 and UGT40A1 proteins,and the differential expression changes of three UGT genes after feeding with different anti-nutritional factors were studied.Our results are expected to provide a reference for the future study of UGT gene functions in silkworm midgut and silkworm artificial feed formula.The main research results are as follows :1.A total of 543048 total spectra were obtained and 37893 peptides and 5468 proteins were identified by analyzing the proteomics differences between artificial diet rearing and mulberry leaf rearing on the fourth day of the fifth instar.A total of 564 up-regulated proteins and 400 down-regulated proteins were identified in the artificial diet group and mulberry leaf group,respectively.GO functional analysis showed that differential proteins were mainly involved in biological processes such as ion binding,oxygen-containing acid metabolism and organic acid metabolism.KEGG pathway analysis showed that it was mainly involved in metabolic pathways,phagocytosis,propionic acid metabolism and galactose metabolism.These results indicated that dietary differences could significantly affect the expression of digestive,metabolic and immune-related proteins in the midgut of silkworm.q RT-PCR analysis showed that the expression of nine genes such as UGT40B4,UGT340C2 and UGT40A1 at the transcriptional level was consistent with the trend of proteome sequencing.2.Bioinformatics analysis of UGT40B4,UGT340C2 and UGT40A1 proteins showed that they had typical hydrophobic regions and typical transmembrane domains.Among them,UGT40A1 protein had two typical hydrophobic regions and two typical transmembrane domains.Homologous protein alignment and phylogenetic analysis revealed that Bombyx mori UGT40A1 protein had the closest relationship with Bombyx mandarina UGT2C1 protein.The UGT40B4 protein of Bombyx mori had the closest genetic relationship with the UGT340C2 protein of Bombyx mori,and had the closest genetic relationship with the UGT340C1.3.q RT-PCR was used to analyze the instar expression profile and tissue expression profile of Bombyx mori.In the instar expression profile,the relative expression levels of UGT40B4 gene in the 1st and 4th instars of Bombyx mori were higher,the relative expression levels of UGT340C2 gene in the 2nd and 3rd instars of Bombyx mori were higher,and the relative expression levels of UGT40A1 gene in the 4th and 5th instars of Bombyx mori were higher.The relative expression level of UGT40B4 gene in Malpighian tubules and midgut of silkworm was higher,indicating that UGT40B4 gene may be related to digestion and metabolism of silkworm.UGT340C2 gene was highly expressed in the epidermis and midgut of Bombyx mori,which may be related to digestive metabolism and epidermis construction.UGT40A1 gene is highly expressed in epidermis,Malpighian tubules,silk gland and head of Bombyx mori,which may have multiple biological functions.4.Autodock Vina software was used for protein-compound molecular docking.The results showed that the binding abilities of UGT40B4,UGT340C2 and UGT40A1 proteins to isoflavones,tannic acid and arabinoxylan were strong,and there were stable hydrogen bonds,which could form stable binding and had the theoretical premise of interaction.5.The feeding experiment of silkworm with soybean isoflavones,tannic acid and arabinoxylan showed that the three UGT genes were highly expressed in the experimental group with soybean isoflavones,which might be related to the metabolic process of soybean isoflavones.Tannic acid can induce UGT340C2 gene expression,suggesting that it is related to tannic acid metabolism.UGT340C2 and UGT40A1 proteins were highly expressed in the experimental group added with arabinoxylans,which may be related to the metabolic process of arabinoxylans.6.Adding 0.1 % and 0.4 % soybean isoflavones in semi-synthetic diets reduced the body weight of silkworm at full-eating stage and affected its growth and development;compared with the control group,the pupal weight of the test group added with soybean isoflavones was significantly increased,indicating that the addition of soybean isoflavones in feed could increase the pupal weight.The investigation of silkworm body weight and cocoon quality during the peak feeding period showed that tannic acid in artificial diet and mulberry leaves had adverse effects on the growth and development of silkworm and the cocooning process of silkworm.Appropriate amount of arabinoxylan had beneficial effects on the growth and cocoon quality of 5th instar silkworm.High content of arabinoxylans will have toxic effects on silkworm,affecting the growth and development of silkworm.Based on the results of this study,soybean isoflavones,tannic acid and arabinoxylan in artificial diets were the reasons for the significant differences in the expression of UGT40B4,UGT340C2 and UGT40A1 proteins in the midgut proteomics of silkworm fed with artificial diets and mulberry leaves.These three anti-nutritional factors have different effects on the growth and development of silkworm.It is speculated that these three UGT proteins are involved in the metabolism of soybean isoflavones,tannic acid and arabinoxylans in the midgut of silkworm,and the metabolic mechanism needs further study.