Open Access Short Research Article

Gene Clone and Bioinformatics Analysis of Subtilisin-Like Protease in Cucumis sativus

Xue Wang, Guangchao Yu, Xiangyu Wang, Chunmao Lv

Biotechnology Journal International, Page 1-9
DOI: 10.9734/bji/2019/v23i130071

Background: Cucumber target leaf spot (TLS), caused by Corynespora cassiicola (C. Cassiicola), is a serious disease in cucumber (Cucumis sativus) production worldwide. Therefore, cultivating new varieties of TLS resistance of C. sativus is an important goal of cucumber breeding. Previous studies have shown that subtilisin-like protease (SUBP) plays an important role in response to C. Cassiicola infection in resistant plants.

Objective: In this study, the full-length cDNA of the CsSUBP gene was cloned, and the prokaryotic expression vector was successfully constructed in order to study the effects of subtilisin. Futhermore, vital clues regarding CsSUBP gene involved in TLS resistance of C. sativus are gained from the bioinformatics assay.

Method: The CsSUBP gene was identified by sequencing with the intermediate vector pMD18 by designing specific primers and PCR amplification techniques. The prokaryotic expression vector pET30a-CsSUBP was further constructed and identified by colony PCR and EcoR V and SalⅠ double digestion.

Result: The primary structure of CsSUBP was predicted and analyzed by bioinformatics analysis. The results showed that CsSUBP was weakly acidic protein, N-terminal signal peptide region, including a Inhibitor_I9 domain domain.

Conclusion: The pET30a-CsSUBP prokaryotic expression vector was constructed successfully. This study is convenient for the study of prokaryotic expression and its kinase activity.

Open Access Original Research Article

Screening and Characterisation of Keratin-Degrading Bacillus sp. from Feather Waste

M. T. Dada, S. M. Wakil

Biotechnology Journal International, Page 1-12
DOI: 10.9734/bji/2019/v23i130067

Aim: This study focuses on the screening and characterisation of keratin-degrading Bacillus species from feather waste.

Methods: Nine bacteria were isolated from feather waste obtained from a poultry layout at Egbeda local government secretariat, Ibadan, Nigeria. These bacteria were grown in basal medium with feather as primary source of carbon, nitrogen, sulfur and energy. Feather degrading bacteria were screened for both proteolytic activity and keratin degradation on skimmed milk agar and keratin azure medium respectively. They were also screened for their ability to degrade other keratin substrates such as hair and nail.

Results: Three of the isolates with higher feather degradation levels also showed high proteolytic activity and release of azure dye. They were selected and identified phenotypically and genotypically using 16S rRNA sequencing as Bacillus licheniformis-K51, Bacillus subtilis-K50 and Bacillus sp.-K53. The bacteria were capable of degrading other keratin-containing substrates such as nail and hair. Bacillus subtilis-K50 and Bacillus licheniformis-K51 showed significant difference (P) in degradation among the three different keratin sources used yielding higher degradation with feather as keratin source with respective optical densities of 0.07 and 0.11 followed by hair and least in nails with optical densities of 0.05 and 0.07 respectively. Highest degradation of all the three keratin substrates was observed in Bacillus licheniformis-K51.

Conclusion: The three isolated bacteria possess the ability to degrade keratin and utilize feather as keratin substrate. As a result, these can be considered as potential candidates for degradation and utilization of feather keratin.

Open Access Original Research Article

Potentials of Sugarcane Bagasse and Poultry Manure in the Remediation of Spent Motor Oil Contaminated Soil

A. Y. Sangodoyin, I. O. Igbode

Biotechnology Journal International, Page 1-11
DOI: 10.9734/bji/2019/v23i130068

This study sought to investigate the potential of sugarcane bagasse, poultry manure and their combination in the remediation of spent motor oil contaminated soil. About 1.5 L of spent motor oil was sprinkled on plots measuring 0.5 m x 0.5 m dimension in 12 replicate plots to simulate a major spill. Poultry dung (PD), Sugarcane bagasse (SB) and hybrid of bagasse and Poultry dung (SB - PD) were applied as organic amendments and bulking agents. The amendments were randomly administered twice within two weeks to treatment plots except the control at rate of 24tons/ha. The soil was tilled to enhance aeration and watered twice a week for 56 days. A significant decrease in Total Petroleum Hydrocarbon, TPH of 61, 53, 46 and 23% for PD, SB - PD, SB and Control plots respectively was observed. TOC and TN decreased in all the plots except the control. Soil moisture contents between 21 and 24% while soil pH within the range of 6.9 to 7.5 enhanced biodegradation process. The Total Heterotrophic Bacterial Counts (THBC) varied in all the plots while heavy metals content remained unchanged during the study. The application of these biostimulants gave promising results on hydrocarbons removal from contaminated soil.

Open Access Original Research Article

The Effect of Nigella sativa and Piper guineense Aqueous Extracts on the in Vivo Expression Profile of Lipid and Sex Hormones

Okoye Ngozi Franca, Uvbiekpahor Rume

Biotechnology Journal International, Page 1-14
DOI: 10.9734/bji/2019/v23i130069

Aim: The study investigated the effect of aqueous extract of Piper guineense and Nigella sativa on Testosterone, Estrogen and Follicle stimulating hormone levels in male and female rats induced by diets supplemented with high dose of sucrose and margarine for four (4) week.

Materials and Methods: Thirty male and female Wistar rats of average weight (100 g) were used for the study. The rats were arranged into five groups with six rats in each of the groups. The rats had access to their normal feed but sucrose and margarine were used to induce hyperglycemia and hyperlipidemia respectively on the rats with the exception of the rats in the positive control group.  The rats in the negative control were induced using the sucrose and margarine but were not treated using the aqueous extracts. The rats in the Piper guineense group were treated with 2 ml of Piper guineense aqueous leaf extract, while the rats in the Nigella sativa group were treated with 2 ml of Nigella sativa aqueous extract. The rats in the Nigella sativa and Piper guineense group were treated with 2 ml of the combined aqueous extract.

Results: The results showed that the extracts had an increasing effect which was time dependent on the hormones. The highest increase was obtained on the third week of feeding when compared to the control. The testosterone levels (mlU/ml) showed for the, positive control (2.28 ± 0.08), uziza leaf (6.45 ± 0.01), black seed (2.85 ± 0.01), black seed &uziza (8.47 ± 0.08)(p<0.05).  The increase for estrogen levels (mlU/ml) showed for the positive control (99 ± 0.82), uziza leaf (98 ± 1.63), black seed (100 ± 1.63), black seed & uziza (113 ± 0.82). Furthermore, the extracts also had an increasing effect on the follicle stimulating hormone levels with the highest increase obtained on the third week (p>0.05).  The FSH levels (mlU/ml)) showed for the positive control (0.23 ± 0.01), uziza leaf 0.25 ± 0.01), black seed (0.54 ± 0.02), black seed & uziza (0.58 ± 0.01)(p<0.05).

Histopathological findings also suggest that treatment with the aqueous extracts of P.guineense and N .sativa after inducement salvaged the testes and ovaries when compared to the induced group after the second week of administration. Although the combined mixture N.sativa and P.guineense extract was more effective.

Conclusion: The results of the study indicate that the aqueous extract of a combined mixture of Nigella sativa and Piper guineense might be an effective plant in indigenous medicine, which may be used to combat infertility caused by hormonal imbalance.

Open Access Original Research Article

Plant Regeneration via Somatic Embryogenesis in Solanum nigrum L. (Black nightshade) (Solanaceae)

Durgam Sharada, Porandla Sai Krishna, Nanna Rama Swamy

Biotechnology Journal International, Page 1-9
DOI: 10.9734/bji/2019/v23i130070

Aim: To study the effect of various plant growth regulators (PGRs) for induction of somatic embryogenesis and plantlet formation from cotyledon and leaflet explants in S. nigrum (night shade) an important medicinal plant used in treatment of digestive problems and skin infections.

Place and Duration of Study: Department of Biotechnology, Kakatiya university, Warangal. Telangana, India, 3 years.

Methodology: Cotyledon (0.8 cm2) and leaflet explants (0.8-1.0 cm2) from 3 week and 4 week old were cultured on MS medium supplemented with 30 g/L sucrose along with different concentrations of 0.5 mg/L BAP+NAA (0.5 – 6.0 mg/L) .

Results: Maximum percentage of somatic embryogenesis was observed in cotyledon(89%) and leaf (98%)  explants on MS medium augmented with  0.5mg/L BAP in combination with  2.0 mg/L NAA whereas the highest number of somatic embryos per explant (86 ± 0.19) was formed in leaflet explant.

Conclusion: Somatic embryogenesis was induced from both cotyledon and leaf explants. Since it is threatened and medicinally important species S. nigrum, the present protocol can be used for its conservation and genetic transformation experiments.