Characterization of Matrix Metalloprotease-9 Gene from Nile tilapia (Oreochromis niloticus) and Its High-Level Expression Induced by the Streptococcus agalactiae Challenge.

The bacterial diseases of tilapia caused by Streptococcus agalactiae have resulted in the high mortality and huge economic loss in the tilapia industry. Matrix metalloproteinase-9 (MMP-9) may play an important role in fighting infection. However, the role of MMP-9 in Nile tilapia against S. agalactiae is still unclear. In this work, MMP-9 cDNA of Nile tilapia (NtMMP-9) has been cloned and characterized. NtMMP-9 has 2043 bp and encodes a putative protein of 680 amino acids. NtMMP-9 contains the conserved domains interacting with decorin and inhibitors via binding forces compared to those in other teleosts. Quantitative real-time-polymerase chain reaction (qPCR) analysis reveals that NtMMP-9 distinctly upregulated following S. agalactiae infection in a tissue- and time-dependent response pattern, and the tissues, including liver, spleen, and intestines, are the major organs against a S. agalactiae infection. Besides, the proteolytic activity of NtMMP-9 is also confirmed by heterologous expression and zymography, which proves the active function of NtMMP-9 interacting with other factors. The findings indicate that NtMMP-9 was involved in immune responses against the bacterial challenge at the transcriptional level. Further work will focus on the molecular mechanisms of NtMMP-9 to respond and modulate the signaling pathways in Nile tilapia against S. agalactiae invasion and the development of NtMMP-9-related predictive biomarkers or vaccines for preventing bacterial infection in the tilapia industry.

Molecular cloning and functional characterization of cathepsin B from Nile tilapia (Oreochromis niloticus).

Cathepsin B (CatB) has been widely known for its hydrolytic ability and involvement in the innate immunity. However, the mechanism of CatB from teleosts participating in immunoregulation remains poorly understood; and the sequence of CatB from Nile tilapia (NtCatB) has not been cloned and characterized. In this study, the coding sequence of NtCatB was cloned, and then characterized by bioinformatic analysis and heterologous expression. The deduced amino acid sequence (330-aa) of NtCatB contains the representative features of CatB. Quantitative real-time PCR revealed the extensive mRNA expression of NtCatB in six tissues of healthy Nile tilapia, and its transcription level was significantly up-regulated after Streptococcus agalactiae challenge. NtCatB may interact with some immunological function proteins and take part in the regulatory pathway. These results suggest that NtCatB is likely to be involved in the immune reaction. The mature region (residues 79-328, mNtCatB) of NtCatB was cloned and transferred to pET-28a for expressing the recombinant protein. The purified recombinant mNtCatB was verified with the activity of 992.34 U mg-1 min-1 under the optimal condition using a substrate hydrolyzing assay. The recombinant cystatin-A1-like can effectively inhibit the activity of the recombinant mNtCatB, and their binding form was predicted by molecular docking. Our results contribute to elucidating the immunological functions of NtCatB.

Molecular characterization and gene expression of cathepsin L in Nile tilapia (Oreochromis niloticus).

Cathepsin L (CatL) has been widely known for its involvement in the innate immunity. However, it still remains poorly understand how CatL modulates the immune system of teleosts. Moreover, the CatL of Nile tilapia (NtCatL) has not been cloned or characterized. In this study, the gene encoding NtCatL was cloned, and was characterized by bioinformatics analysis, heterologous expression and protease activity assay. The coding sequence of NtCatL is 1017 bp in length and encodes 338 amino acid residues with a predicted molecular weight of 38.487 kDa and a theoretical isoelectric point of 5.79. NtCatL possesses the features of a typical cathepsin L, including one signal peptide, one propeptide region, and one papain family cysteine protease domain containing four active site residues (Gln135, Cys141, His281, and Asn305). The prediction of protein-protein interaction shows that NtCatL may interact with some functional proteins for realizing an immune function. Real-time quantitative PCR revealed the widespread transcriptional expression of NtCatL in six tissues of healthy Nile tilapia, and the NtCatL mRNA is significantly up-regulated after Streptococcus agalactiae challenge. These results suggest that NtCatL is likely to be involved in the immune reaction of Nile tilapia. Recombinant proteins from the mature domain (residues 117-337) of NtCatL were obtained by heterologous expression using pET28a and Rosetta (DE3) competent cells. A protein product with the high purity was obtained by using TALON Superflow purification rather than adopting HisTrap HP columns. The protease activity of the recombinant protein was verified by using a substrate hydrolyzing assay. This work has cloned and characterized the CatL from Nile tilapia for the first time, and contributes to elucidating the immunological functions of CatL.

Morphological Observations and Fatty Acid Composition of Indoor-Cultivated Cordyceps sinensis at a High-Altitude Laboratory on Sejila Mountain, Tibet.

Cordyceps sinensis, a caterpillar entomopathogenic fungus-host larva complex, is a rare medicinal herb found in the Qinghai-Tibetan Plateau and its surrounding high-altitude areas. The alternation of generations in the life cycle, whatever the fungus or its host insect, requires special growth conditions. However, it is difficult to simulate the growth conditions of C. sinensis, which hinders its artificial cultivation. In this work, the life cycle from the host larva to C. sinensis was observed in an indoor-cultivation laboratory at 4,200 m a.s.l. on Sejila Mountain, Tibet. Comparative examinations between indoor-cultivated and wild C. sinensis demonstrated that the indoor-cultivated C. sinensis preferred to germinate multiple long, slim stromata at diverse positions on dead larvae, including but not limited to their heads. Their fatty acid composition shows a significant difference in the levels of polyunsaturated fatty acids (PUFAs). In indoor-cultivated C. sinensis, PUFAs constituted 24.59% and 49.43%, respectively, of neutral and polar lipids; meanwhile, in wild C. sinensis, PUFAs represented 34.34% and 61.25% of neutral and polar lipids, respectively. These observations and fatty acid data suggest that environmental factors, particularly temperature, soil pressure and light intensity, strongly affect the growth of C. sinensis. Our new findings may provide important information for improving techniques for the large-scale artificial cultivation of C. sinensis.

Isolation and characterization of a novel hydrocarbon-degrading bacterium Achromobacter sp. HZ01 from the crude oil-contaminated seawater at the Daya Bay, southern China.

Microorganisms play an important role in the biodegradation of petroleum contaminants, which have attracted great concern due to their persistent toxicity and difficult biodegradation. In this paper, a novel hydrocarbon-degrading bacterium HZ01 was isolated from the crude oil-contaminated seawater at the Daya Bay, South China Sea, and identified as Achromobacter sp. Under the conditions of pH 7.0, NaCl 3% (w/v), temperature 28 °C and rotary speed 150 rpm, its degradability of the total n-alkanes reached up to 96.6% after 10 days of incubation for the evaporated diesel oil. Furthermore, Achromobacter sp. HZ01 could effectively utilize polycyclic aromatic hydrocarbons (PAHs) as its sole carbon source, and could remove anthracene, phenanthrene and pyrence about 29.8%, 50.6% and 38.4% respectively after 30 days of incubation. Therefore, Achromobacter sp. HZ01 may employed as an excellent degrader to develop one cost-effective and eco-friendly method for the bioremediation of marine environments polluted by crude oil.