A novel chondroitin AC lyase from Pedobacter xixiisoli: Cloning, expression, characterization and the application in the preparation of oligosaccharides.

Chondroitin AC lyase can efficiently hydrolyze chondroitin sulfate (CS) to low molecule weight chondroitin sulfate, which has been widely used in clinical therapy, including anti-tumor, anti-oxidation, hypolipidemic, and anti-inflammatory. In this work, a novel chondroitin AC lyase from Pedobacter xixiisoli (PxchonAC) was cloned and overexpressed in Escherichia coli BL21 (DE3). The characterization of PxchonAC showed that it has specific activities on chondroitin sulfate A, Chondroitin sulfate C and hyaluronic acid with 428.77, 270.57, and 136.06 U mg-1, respectively. The Km and Vmax of PxchonAC were 0.61 mg mL-1 and 670.18 U mg-1 using chondroitin sulfate A as the substrate. The enzyme had a half-life of roughly 660 min at 37 °C in the presence of Ca2+ and remained a residual activity of 54 % after incubated at 4 °C for 25 days. Molecular docking revealed that Asn123, His223, Tyr232, Arg286, Arg290, Asn372, and Glu374 were mainly involved in the substrate binding. The enzymatic hydrolysis product was analyzed by gel permeation chromatography, demonstrating PxchonAC could hydrolyze CS efficiently.

Molecular characterization and functional analysis of BdFoxO gene in the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

The forkhead box O transcription factor (FoxO) is an important downstream transcription factor in the well-conserved insulin signaling pathway, which regulates the body size and development of insects. In this study, the FoxO gene (BdFoxO) was identified from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frame of BdFoxO (2732 bp) encoded a 910 amino acid protein, and the sequence was well conserved with other insect species. The BdFoxO was highly expressed in larvae and pupae among different development stages, and the highest tissue-specific expression level was found in the fat bodies compared to the testis, ovary, head, thorax, midgut, and Malpighian tubules of adults. Interestingly, we found BdFoxO expression was also up-regulated by starvation, but down-regulated when re-fed. Moreover, the injection of BdFoxO double-stranded RNAs into third-instar larvae significantly reduced BdFoxO transcript levels, which in turn down-regulated the expression of other four genes in the insulin signaling pathway. The silencing of BdFoxO resulted in delayed pupation, and the insect body weight increased significantly compared with that of the control. These results suggested that BdFoxO plays an important role in body size and development in B. dorsalis.

Two Chitin Biosynthesis Pathway Genes in Bactrocera dorsalis (Diptera: Tephritidae): Molecular Characteristics, Expression Patterns, and Roles in Larval-Pupal Transition.

Glucose-6-phosphate isomerase (G6PI) and UDP-N-acetylglucosamine pyrophosphorylase (UAP), two key components in the chitin biosynthesis pathway, are critical for insect growth and metamorphosis. In this study, we identified the genes BdG6PI and BdUAP from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frames (ORFs) of BdG6PI (1,491 bp) and BdUAP (1,677 bp) encoded 496 and 558 amino acid residues, respectively. Multiple sequence alignments showed that BdG6PI and BdUAP had high amino acid sequence identity with other insect homologues. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated that BdG6PI was mainly expressed in the early stages of third-instar larvae and adults, while significantly higher expression of BdUAP was observed in adults. Both transcripts were expressed highly in the Malpighian tubules, but only slightly in the tracheae. The expression of both BdG6PI and BdUAP was significantly up-regulated by 20-hydroxyecdysone exposure and down-regulated by starvation. Moreover, injection of double-stranded RNAs of BdG6PI and BdUAP into third-instar larvae significantly reduced the corresponding gene expressions. Additionally, silencing of BdUAP resulted in 65% death and abnormal phenotypes of larvae, while silencing of BdG6PI had a slight effect on insect molting. These findings provide some data on the roles of BdG6PI and BdUAP in B. dorsalis and demonstrate the potential role for BdUAP in larval-pupal transition.

Insulin signaling pathway in the oriental fruit fly: The role of insulin receptor substrate in ovarian development.

Insulin signaling pathways have integral roles in regulating organ growth and body size of insects. Here, we identified and characterized six insulin signaling pathway components-InR, IRS, PI3K92E, PI3K21B, Akt, and PDK-from Bactrocera dorsalis. Quantitative real-time polymerase chain reaction was used to establish gene expression profiles for the insulin signaling pathway components for different developmental stages and tissues, and in response to 20-hydroxyecdysone (20E) and starvation. IRS, PI3K92E, and PI3K21B were highly expressed in the head, while InR, Akt, and PDK were most abundant in Malpighian tubules. Both IRS and PI3K92E were highly expressed during the larval-pupal and pupal-adult transition, while the remaining four genes were highly expressed only during the pupal-adult transition. Following initial exposure to 20E, the expression levels of most genes were significantly decreased. However, the expression levels of IRS, PI3K92E, and PI3K21B were significantly increased at 8 and 12h post-treatment compared with the control. Moreover, we found that most insulin signaling pathway genes in B. dorsalis were up-regulated in response to starvation, but decreased when re-fed. On the contrary, transcript levels of PI3K21B decreased significantly during starvation. Furthermore, injection of IRS dsRNA into adult females significantly reduced IRS transcript levels. Suppression of IRS expression inhibited ovarian development, and the average ovary size was reduced by 33% compared with the control. This study provides new insight into the roles of insulin signaling pathway components in B. dorsalis, and demonstrates an important role for IRS in ovarian development.