Identification and functional analysis of carbonyl reductases related to tetrahydrobiopterin synthesis in the silkworm, Bombyx mori.

The superfamily of short-chain dehydrogenases/reductases (SDRs) is crucial in biosynthetic and signalling pathways, in which the carbonyl reductases (CBRs) subfamily is important in the biosynthesis of tetrahydrobiopterin (BH4). BH4 is an essential coenzyme for animals, and its deficiency can lead to neurological diseases. There are few reports on CBRs involved in BH4 synthesis of silkworms, Bombyx mori. Here, we identified 67 SDR genes in B. mori (BmSDR) through whole genome survey for the first time. Based on bioinformatics analyses and KEGG verification, four BmCBRs that may be related to BH4 synthesis were further characterized and functionally analysed. The results showed these four genes were high expressed in the head and gonads of ah09 (a lem mutant with defective BH4 synthesis). Enzyme activity, BH4 content and the related gene expression levels after intracellular interference with BmCBR and the main catalytic enzymes sepiapterin reductase of B. mori (BmSpr) in the de novo pathway of BH4 showed BmCBR2 plays a role in the salvage pathway. BmCBR3 and BmCBR4 regulate BH4 synthesis through the alternative pathway. Among the four pathways of silkworm BH4 synthesis, the de novo pathway occupies the dominant position, followed by the alternative pathway and salvage pathway. According to the overexpression of BmCBR3 after interference with BmSpr, the BH4 content did not change significantly. It is speculated that BmCBR3 is located upstream of BmSpr. These results provide a theoretical basis for in-depth exploration of the role of BmSDR in B. mori and also provide clues for the research of other animal-related diseases.

BmSuc1 Affects Silk Properties by Acting on Sericin1 in Bombyx mori.

BmSuc1, a novel animal-type ß-fructofuranosidase (ß-FFase, EC 3.2.1.26) encoding gene, was cloned and identified for the first time in the silkworm, Bombyx mori. BmSuc1 was specifically and highly expressed in the midgut and silk gland of Bombyx mori. Until now, the function of BmSuc1 in the silk gland was unclear. In this study, it was found that the expression changes of BmSuc1 in the fifth instar silk gland were consistent with the growth rate of the silk gland. Next, with the aid of the CRISPR/Cas9 system, the BmSuc1 locus was genetically mutated, and homozygous mutant silkworm strains with truncated ß-FFase (BmSUC1) proteins were established. BmSuc1 mutant larvae exhibited stunted growth and decreased body weight. Interestingly, the molecular weight of part of Sericin1 (Ser1) in the silk gland of the mutant silkworms was reduced. The knockout of BmSuc1 reduced the sericin content in the silkworm cocoon shell, and the mechanical properties of the mutant line silk fibers were also negatively affected. These results reveal that BmSUC1 is involved in the synthesis of Ser1 protein in silk glands and helps to maintain the homeostasis of silk protein content in silk fibers and the mechanical properties of silk fibers, laying a foundation for the study of BmSUC1 regulation of silk protein synthesis in silk glands.

[Xerophthalmia of qi and yin deficiency treated with jinhuobu needling technique].

OBJECTIVE: To explore the differences in the clinical therapeutic effects on xerophthalmia of qi and yin deficiency between the jinhuobu needling technique (the combined reinforcing technique to induce warm sensation) and the reinforcing needing technique achieved by rotating needle. METHODS: A total of 54 patients of xerophthalmia were randomized into an observation group (28 cases) and a control group (26 cases). In both of the groups, the main acupoints included Cuanzu (BL 2), Sizhukong (TE 23), Taiyang (EX-HN 5) and Fengchi (GB 20); the combined acupoints were Danzhong (CV 17), Sanyinjiao (SP 6), Taixi (KI 3) and Taichong (LR 3). In the observation group, the jinhuobu needling technique was used and in the control group, the reinforcing needing technique achieved by rotating needle was adopted. The treatment was given once every day. 10 treatments made one course and 2 courses were required. Before and after treatment, the score of eye symptoms, tear secretion and the breakup time of tear film were observed in the two groups. The clinical therapeutic effects were compared between the two groups after treatment and in 4-week follow-up after treatment separately. RESULTS: After treatment, the total effective rate was 92.9% (26/28) in the observation group and was 80.8% (21/26) in the control group, without significant difference in comparison (P>0.05). In 4-week follow-up after treatment, the total effective rate was 85.7% (24/28) in the observation group and was 61.5% (16/26) in the control group, indicating the significant difference in comparison (P<0.05). After treatment, the differences were significant in the score of eye symptoms, tear secretion and the breakup time of tear film as compared with those before treatment in the two groups (all P<0.01). After treatment, the improvements of the score of eye symptoms, tear secretion and the breakup time of tear film in the observation group were better remarkably than those in the control group, indicating the significant differences (all P<0.05). CONCLUSION: The jinhuobu needling technique achieves the significant therapeutic effects on xerophthalmia of qi and yin deficiency and the recurrence rate of the disease is low.