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    • CCT007093 The fat body is one of the largest tissues of

    2022-12-21

    The fat body is one of the largest tissues of Bombyx mori, with components including fat, glycogen, protein and vitamins. The fat body exhibits important physiological functions, not only storing a large number of nutrients but also serving as an important site of biosynthesis and metabolism (Arrese and Soulages, 2010). Additionally, the fat body is an important immune organ and takes part in the detoxification mechanisms of many molecules (Enayati et al., 2005). The silkworm purple quail-like mutant (q-l) exhibits pigmented dots on its epidermis. In addition, q-l shows developmental abnormalities and decreased vitality. Therefore, we asked whether there is a relationship between the decreased vitality of the q-l mutant and decreased fat body metabolism. Hence, the fat body of the q-l mutant was used as material for two-dimensional gel electrophoresis (2-DE) analysis, with the fat body of the wildtype 932VR strain serving as the control. The results showed that the expression level of BmAR in the q-l mutant was significantly lower than in the wildtype, which remained the same at the RNA level, as verified by quantitative reverse transcription PCR (qRT-PCR). RNAi experiments and determination of sorbitol concentrations indicated that downregulation of BmAR may be related to the developmental abnormality phenotype of the q-l mutant (Zhao et al., 2014). These results provide experimental evidences related to studying the functions of AR in the q-l mutant.
    Materials and methods
    Results
    Discussion In previous research involving transcriptomic analysis of the CCT007093 of the q-l mutant (GEO accession GSE95495), BmAR is also found to be significantly downregulated. The reported RPKM (reads per kb per million reads) value of 932VR is 484.28, while that of q-l is 141.29, and the fold change of q-l relative to 932VR is 0.29 (Wang et al., 2017). The results were consistent with the expression levels of BmAR that we identified in fat bodies at both the protein and mRNA levels. In this study, BmAR was observed to be expressed highly efficiently in all developmental periods and in most tissues, which suggested that BmAR is necessary for silkworm development. Thus, the downregulation of BmAR in q-l might be one of the reasons for the developmental abnormalities of the q-l mutant. One function of AR is transformation of glucose to sorbitol, with the participation of NADPH (Gu et al., 2010; Srivastava et al., 1995). Glucose is a basic substance required by organisms, not only as an important source of energy but also as a synthetic substrate for many important substances as well as the main component of glycoproteins. Additionally, glucose participates in the recognition process (Lairson et al., 2008; Sharon and Lis, 1993). Based on transcriptomic data from the epidermis (GEO accession GSE95495), we analyzed the expression levels of series of key genes in glucose metabolism pathways between wildtype 932VR and q-l silkworms (Table 2). Hexokinase (fold = 0.83) and glucose-6-phosphate isomerase (G6PI, fold = 0.95) are two key enzymes in glycolysis pathway. Hexokinase also catalyzes the first reaction to many other pathways that use glucose as a substrate (Bogorad et al., 2013; Nelson and Cox, 2005). These two enzymes exhibited no difference between 932VR and q-l. The last enzyme in the gluconeogenesis pathway, glucose-6-phosphatase (fold = 0.76) (Nelson and Cox, 2005), also presented no significant difference between 932VR and q-l, which suggested that these two pathways of glucose metabolism were not significantly affected. Glucose-6-phosphate dehydrogenase (G6PD, fold = 0.33) is a key enzyme in the pentose-phosphate pathway that catalyzes the transformation of glucose 6-phosphate to 6-phosphate gluconic acid-δ-lactone (Bogorad et al., 2013; Nelson and Cox, 2005). In q-l, G6PD was downregulated approximately 3-fold, which suggested that the pentose-phosphate pathway might be affected. The synthesis of chitin, an important component of the insect epidermis, also begins with glucose, which is eventually converted to UDP-N-acetylglucosamine (Candy and Kilby, 1962). A series of genes involved in the chitin biosynthesis pathway are also differentially expressed in q-l, resulting in an increased chitin content (Wang et al., 2017). UDP-glycosyltransferases (UGTs) are a superfamily of enzymes that mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones) (Huang et al., 2008). By analyzing the expression levels of UTGs screened from the genome of Bombyx mori (Huang et al., 2008) as well as GO BLAST results for the transcriptome of the epidermis (Wang et al., 2017), we found that 4 UGTs were downregulated (<0.5-fold) in q-l, while 5 were upregulated (>2-fold). These results suggested that the glycosylation process was also affected in the q-l mutant. In conclusion, some glucose-related physiological processes were found to be abnormal in q-l, which might be a factor contributing to the developmental abnormalities of the q-l mutant.