Identification and characteristics of a novel gene, <Emphasis Type="Italic">EJO1</Emphasis>, in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir japonica sinensis</Emphasis>) ovary

EJO1, a novel gene presumably involved in the ovary development of the Chinese mitten crab (Eriocheir japonica sinensis), was identified and characterized by suppression subtractive hybridization and cDNA macroarray analysis. EJO1 expression was 2.6-fold higher at stage III than at stage II during the ovary development of the mitten crab. EJO1 is 876 bp in length containing a 759 bp open reading frame which encodes a 252-amino-acid protein. Homology analysis showed that no sequence significantly matching EJO1 was found in SwissProt, so it was deduced as a novel gene (GenBank accession number: AY185917). The EJO1 protein is probably a secretion protein with a signal peptide of 17 amino acids. The pI/Mw deduced from the amino acid sequence was 6.18/28.18 kDa. Expression profile showed that EJO1 mRNA is highly expressed in the heart, intestine, and ovary of the crab, while there is little or no expression in muscle and hepatopancreas. The differential expression of EJO1 at the different developmental stages of the ovary was further confirmed by Northern blot analysis. In conclusion, EJO1 is a novel gene differentially expressed in the ovary of the Chinese mitten crab, which may play an important role in the ovary development.     

Expression of Protein Complex Comprising the Human Prorenin and (Pro)Renin Receptor in Silkworm Larvae Using <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus (BmNPV) Bacmids for Improving Biological Function

Three forms of recombinant protein complexes comprising the human prorenin (hPro) and (pro)renin receptor (hPRR) (hPRR/prorenin) were successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids. They were localized in the fat body cells and formed a prorenin-bound hPRR complex. The expressed levels of hPro and hPRR were similar judging from Western blotting. The hPRR/prorenin complex containing 40 μg of hPRR (yield, 43%) and 30 μg of hPro (yield, 34%) was purified from 15 silkworm larvae by a series of purification using anti-FLAG and Strep-Tactin affinity chromatography. The renin activity of the purified hPRR/prorenin complex was 3.8-fold that of the mixture of hPRR and hPro expressed individually in vitro judging from the renin assay. These results show that the unstable transmembrane protein, hPRR, was coexpressed stably with ligand, hPro, and formed a stable protein, hPRR/prorenin complex that showed a high catalytic active form.     

Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector

The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human granucyto-macrophage colony-stimulating factor (hGM-CSF) in the posterior silk glands of gene-targeted silkworms, a targeting vector pSK-FibL-L-A3GFP-PH-GMCSF-LPA-FibL-R was constructed, harboring a 1.2 kb portion of the left homogenous arm (FibL-L), a 0.5 kb portion of the right homogenous arm (FibL-R), fibroin H-chain-promoter-driven hGM-CSF and silkworm actin 3-promoter-driven gfp. The targeting vector was then introduced into the eggs of silkworm, and the transgenic silkworms were verified by PCR and DNA hybridization after being screened for the gfp gene. Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kD in the silk glands of the G3 generation transgenic silkworms. The level of expression of hGM-CSF in the posterior silk glands of the G3 generation transgenic silkworms was approximately 2.70 ng/g of freeze-dried powdered posterior silk gland. These results showed that the heterologous gene could be introduced into the silkworm genome and expressed successfully. Further more, the exogenous genes existing in the G5 transgenic silkworm identified by PCR confirmed its integration stability. In addition, the silk glands containing expressed hGM-CSF performed the function of significantly increasing leukocyte count of CY-treated mice in a time-and-dose-dependent manner.     

Protein Profile of <Emphasis Type="Italic">Nomuraea rileyi</Emphasis> Spore Isolated from Infected Silkworm

Nomuraea rileyi (N. rileyi) is the causative agent of the silkworm, Bombyx mori, green muscardine which can cause severe worldwide economical loss in sericulture. Little is known about N. rileyi at the protein level for this entomopathogenic parasite which belongs to the Ascomycota. Here, we employed proteomic-based approach to identify proteins of N. rileyi spores collected from the dead silkworm. In all, 252 proteins were separated by two-dimensional gel electrophoresis (2-DE), and were subjected to mass spectrometry (MS) analysis, 121 proteins have good MS signal, and 24 of them were identified due to unavailability of genomic information from N. rileyi. This data will be helpful in understanding the biochemistry of N. rileyi.     

Overproduction of soluble recombinant transglutaminase from <Emphasis Type="Italic">Streptomyces netropsis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed activity equivalent to the authentic mature TGase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bacterium‐Expressed dsRNA Downregulates Microsporidia Nosema bombycis Gene Expression

The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain (DE3) to express double‐strand RNAs targeting the gene encoding ADP/ATP protein in N. bombycis. The results showed that dsRNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the dsRNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm.     

Expression of <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> endoglucanase III in the larvae of silkworm, <Emphasis Type="Italic">Bombyx mori L.</Emphasis> and characteristic analysis of the recombinant protein

Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell line and silkworm larvae with an improved Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the chiA and v-cath genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The result showed that around 45 kDa protein was visualized in BmN cells at 48 h after the second generation recombinant mBacmid/BmNPV/EG III baculovirus infection. The enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 8.0 and temperature 50°C, and increased 20.94 and 19.13% compared with that from blank mBacmid/BmNPV baculoviruses infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 9.0 and at temperature range from 40 to 60°C. It provided a possibility to generate transgenic silkworms expressing bio-active cellulase, which can catabolize dietary fibers more efficiently, and it might be of great significance for sericulture industry.     

Insight into the biochemical characteristics of a novel glucokinase gene mutation

Glucokinase (GCK) acts as a glucose sensor and regulates β-cell insulin secretion. The heterozygous mutations in the gene encoding GCK cause a reduction of the enzyme activity, which results in a monogenic form of diabetes, maturity-onset diabetes of the young. In the present study, we identified and functionally characterized a novel missense mutation in the GCK gene, which results in a protein mutation Glu³³⁹ → Lys (E339K), from a Chinese family with hyperglycemia. The same GCK mutation that co-segregated with diabetes phenotype was identified in five members of this family but was not found in 200 healthy control individuals. We expressed and affinity-purified the GCK proteins from bacterial expression system that carries mutation (E339K) and fused to glutathione S-transferase. The expressed GCK protein was subjected to the measurement of its biochemical effects of the missense mutation on GCK activity. Our results showed that the mutation reduced the GCK protein yield. The enzymatic kinetics and the thermal stability analysis on the recombinant GCK proteins revealed that the mutation inactivates enzyme kinetics and severely impaired the GCK protein stability.     

Cloning and expression of a novel thermostable cellulase from newly isolated <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain I15

A novel bacterial strain with high cellulase activity (2.82 U/ml) was isolated, and then identified by its morphological character and 16S rRNA sequence, and named Bacillus subtilis strain I15. The extracellular thermostable cellulase exhibited the maximum activity at 60°C and pH 6.0. It was very stable since more than 90% of original CMCase activity was maintained at 65°C after incubation for 2 h. The cellulase gene, celI15, was cloned and extracellularly expressed by Escherichia coli BL21 (DE3), which encoded the extracellular protein of about 52 kDa. The extracellular activity of CelI15 from E. coli BL21 was up to about 6.78 U/ml, and all the other properties were almost the same as that from the wild-type strain.     

Production and characterisation of AoSOX2 from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>, a novel flavin-dependent sulfhydryl oxidase with good pH and temperature stability

Sulfhydryl oxidases have found application in the improvement of both dairy and baking products due to their ability to oxidise thiol groups in small molecules and cysteine residues in proteins. A genome mining study of the available fungal genomes had previously been performed by our group in order to identify novel sulfhydryl oxidases suitable for industrial applications and a representative enzyme was produced, AoSOX1 from Aspergillus oryzae (Faccio et al. BMC Biochem 11:31, 2010). As a result of the study, a second gene coding for a potentially secreted sulfhydryl oxidase, AoSOX2, was identified in the genome of A. oryzae. The protein AoSOX2 was heterologously expressed in Trichoderma reesei and characterised with regard to both biochemical properties as well as preliminary structural analysis. AoSOX2 showed activity on dithiothreitol and glutathione, and to a lesser extent on D/L-cysteine and beta-mercaptoethanol. AoSOX2 was a homodimeric flavin-dependent protein of approximately 78 kDa (monomer 42412 Da) and its secondary structure presents alpha-helical elements. A. oryzae AoSOX2 showed a significant stability to pH and temperature.     

Cloning and expression analysis of a pollen preferential rapid alkalinization factor gene, <Emphasis Type="Italic">BoRALF1</Emphasis>, from broccoli flowers

Rapid alkalinization factors (RALFs) are recently reported active peptide hormones and are considered to play important roles in plant development. We previously identified a differentially expressed cDNA fragment between cabbage flower buds of sterility lines and its maintainer line, which showed significant homology to Arabidopsis RALFL9. The novel RALF cDNA (BoRALF1) was isolated from broccoli flower buds by EST assembly. The open reading frame (ORF) comprises 240 bp, encoding a small putative preprotein of 79 amino acids (molecular weight of 8.72 kDa and a pI of 7.8), which contains the mature polypeptide at its C terminus. BoRALF1 shares 70.3% identity with Arabidopsis RALFL9, but has only moderate similarity with functionally characterized RALFs (ranging from 16.2% to 38.0%). BoRALF1 shows typical features of RALFs, including the 28-aa signal peptide, typical arrangement of four position conserved cysteines, the YIXY motif and a similar secondary structure. RT-PCR studies of different tissues and promoter-GUS fusions confirmed that BoRALF1 is expressed strictly in mature pollen grains and in the anther cells around the loculi. Based on in vivo transient assays, we found that BoRALF1 appears to be largely localized in the plasma membrane. Although the function of BoRALF1 remains to be determined, our experiments confirm the presence of RALF peptide in broccoli, and suggest it could have a role in anther or pollen development.     

Molecular characterization and expression patterns of <Emphasis Type="Italic">Lbx1</Emphasis> in porcine skeletal muscle

Ladybird-like genes were recently identified in mammals. The first member characterized, Lbx1, is expressed in developing skeletal muscle and the nervous system. However, little is known about the porcine Lbx1 gene. In the present study, we cloned and characterized Lbx1 from porcine muscle. RT-PCR analyses showed that Lbx1 was highly expressed in porcine skeletal muscle tissues. And we provide the first evidence that Lbx1 has a certain regulated expression pattern during the postnatal period of the porcine skeletal muscle development. Lbx1 gene expressed at higher levels in biceps femoris muscles compared with masseter, semitendinosus and longissimus dorsi muscles in Meishan pigs. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, single nucleotide polymorphism (SNP) scanning in the Lbx1 genomic fragment identified two mutations, g.752A>G and g.−1559C>G. Association analysis in our experimental pig populations showed that the mutation of g.752A>G was significantly associated with loin muscle area (P < 0.05) and internal fat rate (P < 0.05). Our results suggest that the Lbx1 gene might be a candidate gene of carcass traits and provide useful information for further studies on its roles in porcine skeletal muscle.     

Genome‐wide identification of chitin‐binding proteins and characterization of BmCBP1 in the silkworm,Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.     

Characterization and heterologous expression of a novel lysophospholipase gene from Antrodia cinnamomea

Aims: A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 ‐bp open reading frame encoding 314 amino acids with a molecular weight of 35·5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser–His–Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C‐terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long‐chain acyl esterases at pH 7 and 30°C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4°C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry. Conclusion: We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities. Significance and Impact of the Study: This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level.