Midgut Transcriptome Analysis,Digestion-absorption And Disease Resistance Related Genes Expression In Bombyx Mori Reared With Artificial Diet And Mulberry Leaf

At present,the technology of young silkworm larvae rearing with artificial diet reached the basically practical requirements of silkworm industry,but there are still some problems in artificial diet rearing for all silkworm instars,such as poor resistance,low cocoon shell rate and low feed efficiency.This mode,rearing young silkworm larvae rearing with artificial diet and grown silkworm larvae rearing with mulberry leaf is the developing direction of sericulture in China in the near future,and silkworm rearing with factory artificial diet at all ages is the goal of the development of sericulture in the future.However,there is still a lack of systematic research on the differences and molecular mechanism in digestion-absorption and disease resistance of artificial diet rearing for all instars and mulberry leaf rearing for all instars,and the change from young silkworm artificial diet rearing to grown silkworm mulberry leaf rearing.Therefore,based on the artificial diet rearing and mulberry leaf rearing in the midgut transcriptome sequencing and comparative analysis,we selected 35 differentially expressed genes related to digestion and absorption,defense and energy metabolism.We determined the main digestive enzyme activity and differences in gene expression changes under different rearing.In order to provide reference for revealing the physiological transformation mechanism of digestive tract,improve recipe of artificial diet and rearing technology of silkworm.The main results are as follows:1.In this study,the transcriptome differences in the midgut of silkworm reared with artificial diet and mulberry leaf were analyzed by high-throughput sequencing.A total of12667 expression genes were obtained(|log2FC|≥1,Q≤0.001),including 6512 differentially expressed genes.Among them,the expression levels of 6339 genes in artificial diet were higher than those in mulberry leaf rearing,and the expression levels of 173 genes in mulberry leaf rearing were higher than those in artificial diet rearing.KEGG Pathway analysis showed that the differentially expressed genes involved multiple metabolic pathways.The results of transcriptome analysis preliminarily proved that there were significant differences in digestive absorption,disease resistance,energy metabolism and material transport between artificial diet and mulberry leaf rearing of silkworm in the midgut.2.Biochemical method and qPCR technology were used to determine the activity of representative digestive enzymes and the expression differences of some genes related to digestion and defense in the intestinal fluid of fifth instar larve reared with artificial diet and mulberry leaf.The results showed that the activity of α-amylase and the relative expression levels of amylase gene Amy,trypsin-like gene,serine protease inhibitor gene(serpin-9)and chitinase gene Chi-h in the intestinal fluid of the fifth instar larve reared with artificial diet were significantly higher than those of the mulberry leaf rearing,while the activity and gene expression levels of lipase and serine protease,most resistance-related genes and chitinase genes in the artificial diet rearing were significantly lower than those in the mulberry leaf rearing of silkworm.The relative expression levels of some genes are not consistent with their enzyme activities,and the enzyme activities are also affected by activators and inhibitors in artificial diet.3.Bm NPV was orall inoculated into the second day of the fifth instar silkworms reared with artificial diet and mulberry leaves.The mortality of the former was 100 % and that of the latter was 91.67 %.At 12 h after NPV injection,the activities of serine proteases and lipases with dual functions of digestion and disease resistance in the intestinal fluid of artificial diet and mulberry leaf rearing of silkworm were significantly higher than those in the blank control group,but the expression of chbp and Bmlipase-1 gene and enzyme activity were not the same.Compared with mulberry leaf rearing,there was no uniform regularity in the activity and gene expression of different digestive enzymes.4.It was found that the activities of lipase and serine protease were in the order of bacterial gastroenteritis in silkworms reared with artificial diet > normal silkworm reared with artificial diet > normal silkworm reared with mulberry leaves,while the activities of α-amylase,sucrase and maltose were in the opposite order,indicating that the activity of lipase and serine protease was enhanced by bacterial gastroenteric disease in silkworm,which was consistent with the effect of NPV infection,while the activity of carbohydrate digestive enzymes was reduced.The expression levels of related to immunity and defense in silkworm infected with bacterial gastroenteritis were significantly lower than those in normal silkworm,such as two antimicrobial peptide genes(6tox,Mor),fungal protease inhibitor gene(LOC101738434)and chitin synthase gene(CHS-2),while the expression level of chitinase gene(Chi-h)showed the opposite trend.5.The changes of enzyme activity and gene expression related to midgut digestion and absorption,defense and oxidative phosphorylation in the fifth instar larvae of silkworm were determined after the transformation from artificial diet to mulberry leaf rearing.The results showed that the expression levels of five kinds of digestive enzyme activities,three digestive enzyme genes,seven defense-related genes,three chitin synthesis and degradation-related genes,five energy metabolism-related genes,glycerol and phospholipid metabolism-related genes,and two carboxypeptidase genes were low in the early-stage of the fifth instar,but high in the middle and late stage of the fifth instar,which was suitable for large-scale feeding in the mid-stage of the fifth instar.Among them,for the activity of five digestive enzymes and the relative expression of their genes,the general trend is that the silkworm reared with mulberry leaf all instars is higher than the silkworm reared with artificial diet and the silkworm from artificial diet rearing to mulberry leaf rearing.Two antimicrobial peptide genes and chlorophyll a binding protein gene chbp expression,the overall trend is the artificial diet rearing for all instars test group > 72 h of the fifth instar transform into mulberry leaf rearing test group > newly fourth-exuviated larve transform into mulberry leaf rearing test group,especially in the late-stage of the fifth instar is more obvious,and the expression of AEP gene changes in contrast.Compared with the artificial diet rearing all instars,the expression changes of the above genes in the newly fourth-exuviated larve transform into mulberry leaf rearing test group and 72 h of the fifth instar transform into mulberry leaf rearing test group,there was no uniform regularity.Based on the results of this experiment,the digestive and absorption capacity and defense ability of the midgut of silkworm reared with artificial diet were generally lower than those of silkworm reared with mulberry leaves.Therefore,it is necessary to further improve recipe of artificial diet and strengthen the disease prevention measures.After using the technology of artificial diet rearing of young silkworms and mulberry leaf rearing of grown silkworms,the digestion-absorption and disease-resistance ability of silkworms could not reach the level of mulberry leaf rearing for all instars in a short period of time,so it is necessary to strengthen feeding management and nutrition to achieve high yield and good quality.The adaptation mechanism and regulation mechanism in the process of artificial diet rearing of young silkworm into mulberry leaf rearing of grown silkworm remain to be further studied.