RESUMEN
Sericins are large proteins with molecular weights >70â¯kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280â¯kDa and 260â¯kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks.
Asunto(s)
Adhesivos/química , Adhesivos/metabolismo , Bombyx/genética , Bombyx/metabolismo , Sericinas/química , Sericinas/metabolismo , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Transporte de Proteínas , Sericinas/genética , Seda/metabolismoRESUMEN
Developing a non-toxic, super-absorbent and antibacterial hydrogel as a skin wound dressing is of significant importance. Sericin hydrogel is an ideal dressing material as it has excellent biocompatibility, biodegradability, moisture retention, high affinity to biomolecules, self-healing and promoting cell proliferation activity. Here, we blended silk sericin (SS) with poly(vinyl alcohol) (PVA) to prepare a SS/PVA hydrogel through repetitive freeze-thawing. The photoluminescence of SS/PVA hydrogel indicated PVA was well blended with sericin. SS/PVA hydrogel showed excellent hydrophilicity and swelling behavior for its porous structure. PVA blending effectively improved the thermostability of sericin and enhanced the mechanical property of sericin, but did not affect the crystallinity of sericin and PVA. SS/PVA hydrogel exhibited the ability to load and release small molecule drugs and silver nanoparticles. Cytotoxicity and immuno-toxicity assays suggested the gentamicin loaded SS/PVA hydrogel had excellent cytocompatibility on mammalian cells. Bacterial inhibition assay and wound infection model demonstrated the gentamicin loaded SS/PVA hydrogel could effectively inhibit bacterial growth, thus maintain cells viability. This novel hydrogel with antimicrobial activity has shown great potential in wound dressing.
Asunto(s)
Vendajes , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Hidrogeles/química , Alcohol Polivinílico/química , Sericinas/química , Cicatrización de Heridas , Animales , Antibacterianos/farmacología , Aspirina/farmacología , Bacterias/efectos de los fármacos , Bombyx , Fuerza Compresiva , Liberación de Fármacos , Gentamicinas/farmacología , Células HEK293 , Humanos , Luminiscencia , Ratones , Pruebas de Sensibilidad Microbiana , Células 3T3 NIH , Porosidad , Células RAW 264.7 , Espectroscopía Infrarroja por Transformada de Fourier , Termogravimetría , Agua/química , Difracción de Rayos XRESUMEN
The silk gland of silkworm produces silk proteins during larval development. Many studies have long focused on the silk gland of the fifth instar larvae, but few have investigated this gland at other larval stages. In the present study, the silk gland proteomes of the fourth instar and fourth molt are analyzed using liquid chromatography-tandem mass spectrometry. In total, 2654 proteins are identified from the silk gland. A high abundance of ribosomal proteins and RR-motif chitin-binding proteins is identified during day 2 of the fourth instar (IV-2) larval developmental stage, and the expression of cuticular proteins analogous to peritrophin (CPAP)-motif chitin-binding proteins is higher during the fourth molt (IV-M). In all, nine enzymes are found to be involved in the chitin regeneration pathway in the silk gland. Among them, two chitinase and two chitin deacetylases are identified as CPAP-motif proteins. Furthermore, the expression of CPAP3-G, the most abundant CPAP-motif cuticular protein in the silk gland during the IV-M stage, is investigated using western blot and immunofluorescence analyses; CPAP3-G shows a reverse changing trend with chitin in the silk gland. The findings of this study suggest that CPAP-motif chitin-binding proteins are involved in the degradation of the chitin layer in the silk gland. The data have been deposited to the ProteomeXchange with identifier PXD008677.
Asunto(s)
Bombyx/fisiología , Quitina/metabolismo , Proteínas de Insectos/metabolismo , Proteoma/análisis , Seda/metabolismo , Secuencias de Aminoácidos , Animales , Bombyx/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Insectos/genética , Muda , Dominios Proteicos , RegeneraciónRESUMEN
POU-M2 is a homeodomain transcription factor which plays important roles in the development and silk synthesis of Bombyx mori. In this study, we expressed, purified and characterized POU-M2 and studied its transcription regulation on fibroin heavy chain gene of Bombyx mori. Gel filtration showed POU-M2 existed as a dimer in solution. Far-UV circular dichroism spectra indicated POU-M2 had a well-defined α-helix structure and the α-helix content was about 26.4%. The thermal unfolding transition of POU-M2 was a cooperative process. Tm, ΔH and ΔS were 45.15 ± 0.2 °C, 138.4 ± 0.5 KJ/mol and 0.4349 ± 0.04 KJ/(mol·K), respectively. Western blotting analysis indicated the expression level of POU-M2 increased slightly from day 3 to day 7 of the fifth instar larvae in the posterior silk gland. POU-M2 was positioned in the nucleus of cells. The luciferase reporter assay demonstrated POU-M2 could stimulate the promoter activity of fibroin heavy chain gene, and the activation effect was dependent on the amount of POU-M2. Our study suggested POU-M2 may be involved in the transcriptional regulation of fibroin heavy chain gene. These findings expand toward a better understanding of the structure of POU-M2 and its function in silk synthesis of Bombyx mori.
Asunto(s)
Bombyx/metabolismo , Proteínas de Insectos/metabolismo , Factores del Dominio POU/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bombyx/genética , Biología Computacional , Fibroínas/genética , Regulación de la Expresión Génica , Proteínas de Insectos/química , Proteínas de Insectos/genética , Factores del Dominio POU/química , Factores del Dominio POU/genética , Regiones Promotoras Genéticas/genética , Estructura Secundaria de Proteína , Seda/biosíntesisRESUMEN
Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra. The results showed that NusA and MBP could significantly enhance the soluble expression of Bmsage in E. coli, but it was difficult to separate Bmsage from these tags. SUMO could not only increase the soluble expression of Bmsage in E. coli to a certain degree, but also be effectively separated from Bmsage. Other tags did not effectively promote the soluble expression of Bmsage in E. coli. Circular dichroism spectra showed that the purified Bmsage had well-defined α-helix structure in solution, indicating that SUMO may promote the correct folding of Bmsage into native-like structure. These work not only establish a foundation for further study of the property, structure and function of Bmsage, but also provide a reference for the expression and purification of other similar proteins.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Bombyx , Proteínas de Insectos/biosíntesis , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Escherichia coli , Proteínas de Insectos/química , Estructura Secundaria de Proteína , SedaRESUMEN
Silk proteins are synthesized in the middle and posterior silk glands of silkworms, then transit into the anterior of the silk gland, where the silk fibers are produced, stored and processed. The mechanism of formation and spinning of the silk fibers has not been fully elucidated, and transcriptome analyses specific to the anterior silk gland have not been reported. In the present study, we explored gene expression profiles in five regions of silk gland samples using the RNA-Seq method. As a result, there were 959,979,570 raw reads obtained, of which 583,068,172 reads were mapped to the silkworm genome. A total of 7419 genes were found to be expressed in terms of reads per kilobase of exon model per million mapped reads ≥ 5 in at least one sample. The gene numbers and expression levels of the expressed genes differed between these regions. The differentially expressed genes were analyzed, and 282 genes were detected as up-regulated in the anterior silk gland, compared with the other parts. Functions of these genes were addressed using the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases, and seven key pathways were enriched. It suggested that the ion transportation, energy metabolism, protease inhibitors and cuticle proteins played essential roles in the process of silk formation and spinning in the anterior silk gland. In addition, 210 genes were found differently expressed between males and females, which should help to elucidate the mechanism of the quality difference in silk fibers from male and female silkworms.
Asunto(s)
Bombyx/genética , Perfilación de la Expresión Génica/métodos , Proteínas de Insectos/genética , Seda/metabolismo , Transcriptoma/genética , Animales , Mapeo Cromosómico , Análisis por Conglomerados , Femenino , Ontología de Genes , Genoma de los Insectos/genética , Glucólisis/genética , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Transducción de Señal/genéticaRESUMEN
The partial sequences of exon I of hormone-sensitive lipase (HSL) genes in yak (Bos grunniens), cattle (Bos taurus), zebu (Bos indicus), and buffalo (Bubalus bubalis) were analyzed. Comparisons of these sequences and the deduced amino acid sequences with the homologous HSL gene and protein sequences in other mammalian species including pig (Sus scrofa), human (Homo sapiens), mouse (Mus musculus), and rat (Rattus sp.) retrieved from the GenBank were carried out and finally a phylogenetic tree was constructed using the partial DNA sequences of the HSL genes in all species. The results showed that the homologies of the partial exon I sequences of the HSL genes between yak and cattle, zebu, buffalo, pig, human, mouse, and rat were as high as 99.8%, 99.6%, 97.4%, 90.6%, 88.4%, 83.5%, and 82.3%, respectively. This was accompanied by highly homologous amino acid sequences of the HSLs: 100%, 100%, 98.2%, 94.0%, 92.2%, 89.8%, and 89.8% identity, respectively. There are more transitions, less transversions, and no insertion or deletion in variable nucleotides of the HSL genes between the yak and other species. The majority of the variable mutations was synonymous and was found most frequently at the third codon, followed by the first and second codons, a finding that was in accordance with the neutralism hypothesis for molecular evolution. In the phylogenetic tree, the cattle and zebu were clustered together first, followed by the yak, buffalo, pig, human, mouse, and rat. This was in agreement with taxonomy suggesting that the partial sequences of exon I of the HSL genes were useful in constructing the phylogenetic tree of mammalian species. Among the four species of Bovidae, genetic differentiation in the HSL genes between yak and buffalo is equivalent to that between buffalo and cattle and between buffalo and zebu. Furthermore, the genetic distances in the HSL genes are much smaller between yak, cattle, and zebu than those between each of the three species and the buffalo. Therefore, it is reasonable to consider yak as an independent species of the genus Bos.
Asunto(s)
Bovinos/genética , Evolución Molecular , Variación Genética , Esterol Esterasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Búfalos/genética , Bovinos/clasificación , Exones , Humanos , Mamíferos/clasificación , Mamíferos/genética , Ratones , Datos de Secuencia Molecular , Filogenia , Ratas , Alineación de Secuencia , Esterol Esterasa/químicaRESUMEN
Single nucleotide polymorphisms (SNPs) in partial 5' regulatory region of the insulin-like growth factor 2 (IGF2) gene were studied by DNA sequencing in 60 pigs from the Wuzhishan, Diannan small-ear, Xiang, Meishan and Large White pig breeds. Thirteen SNP sites were detected, including one transversion at T6029A, 4 A<---->G transitions (A5976G, G13520A, G13563A and G13669A) and 8 C<---->T transitions (C5872T, C5888T, C6010T, C6037T, C6043T, C6063T,C6112T, C6164T). These 13 SNPs formed 23 composite genotypes. The gene, genotype and composite genotype frequencies of every SNP site in the whole group and in each breed were calculated. Results showed that the predominant allele in 3 miniature pig breeds was G, T and A at A5976G, C6164T and G13669A sites respectively, but the A-C-G allele was pre-dominant in Meishan and Large White breeds. Moreover, H15 and H19 were the characteristic composite genotype for the large versus the miniature breeds, respectively. In addition, the C5888T SNP was analyzed in 123 Wuzhishan pigs by the PCR-RFLP method. Results showed that the predominant allele was C, and the predominant genotype was CC. chi2-test results indicated that the Wuzhishan pig breed was at Hardy-Weinberg equilibrium with respect to this SNP. These results provide the miniature pig breeds such as the Wuzhishan pig with certain genetic references on the regulation of growth and development, and the mechanism of its dwarfism.