Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 18 num. 2 lang. es <![CDATA[SciELO Logo]]> <![CDATA[<strong>Identification of groundnut (</strong><em><b>Arachis hypogaea</b></em><strong>) SSR markers suitable for multiple resistance traits QTL mapping in African germplasm</strong>]]> Background This study aimed to identify and select informative Simple Sequence Repeat (SSR) markers that may be linked to resistance to important groundnut diseases such as Early Leaf Spot, Groundnut Rosette Disease, rust and aflatoxin contamination. To this end, 799 markers were screened across 16 farmer preferred and other cultivated African groundnut varieties that are routinely used in groundnut improvement, some with known resistance traits. Results The SSR markers amplified 817 loci and were graded on a scale of 1 to 4 according to successful amplification and ease of scoring of amplified alleles. Of these, 376 markers exhibited Polymorphic Information Content (PIC) values ranging from 0.06 to 0.86, with 1476 alleles detected at an average of 3.7 alleles per locus. The remaining 423 markers were either monomorphic or did not work well. The best performing polymorphic markers were subsequently used to construct a dissimilarity matrix that indicated the relatedness of the varieties in order to aid selection of appropriately diverse parents for groundnut improvement. The closest related varieties were MGV5 and ICGV-SM 90704 and most distant were Chalimbana and 47-10. The mean dissimilarity value was 0.51, ranging from 0.34 to 0.66. Discussion Of the 376 informative markers identified in this study, 139 (37%) have previously been mapped to the Arachis genome and can now be employed in Quantitative Trait Loci (QTL) mapping and the additional 237 markers identified can be used to improve the efficiency of introgression of resistance to multiple important biotic constraints into farmer-preferred varieties of Sub-Saharan Africa. <![CDATA[<strong>Inhibitory effect of lotus seedpod oligomeric procyanidins on advanced glycation end product formation in a lactose-lysine model system</strong>]]> Background Industrial food processing induces protein glycation modifications and toxic advanced glycation end products (AGEs) which affect human health. Therefore, it is of interest to monitor AGEs in food processing. The present study was carried out to investigate the influence of lotus seedpod oligomeric procyanidin (LSOPC) concentrations, solution pH value and metal ions on AGE formation by heat treatment of lactose-lysine model solutions. Ne-(carboxymethyl) lysine (CML), as one of the common AGEs was also determined by HPLC-MS/MS in this experiment. Results The results showed that LSOPC can inhibit the formation of AGEs effectively at higher concentrations, lower temperature, and it can reverse the promotion function of metal ions because of its high inhibition activity. Also, LSOPC can inhibit CML formation in the Maillard reaction as well. Conclusion These results indicated that LSOPC could be used as functional food ingredients to inhibit AGE formation. <![CDATA[<strong>Compilation of an informative microsatellite set for genetic characterisation of East African finger millet (</strong><em><b>Eleusine coracana</b></em><strong>)</strong>]]> Background Genetic diversity of finger millet (Eleusine coracana), a nutritious neglected staple cereal in Africa and South Asia is largely uncharacterized. This study analysed 82 published SSR markers for finger millet across 10 diverse accessions to compile an informative set for genetic characterisation. Extensive optimization compared single samples with bulked leaf or bulked DNA samples for capturing within accession genetic diversity. The markers were evaluated to determine (1) how efficiently they amplified target loci during high-throughput genotyping with a generic PCR protocol, (2) ease of scoring PCR products and (3) polymorphism and ability to discern genetic diversity within the tested finger millet germplasm. Results Across 88 samples, the 52 markers that worked well amplified 274 alleles, ranging from 2 to 14 per locus with a mean of 4.89. Major allele frequency ranged from 0.18 to 0.93 with a mean of 0.57. Polymorphic Information Content (PIC) ranged from 0.13 to 0.88 with a mean of 0.5 and availability varied between 64 and 100% with a mean of 92.8%. Heterozygosity ranged from 0 to 1.0, with a mean of 0.26. Discussion Five individual samples from an accession captured the largest number of alleles per locus compared to the four different bulked sampling strategies but this difference was not significant. The identified set comprised 20 markers: UGEP24, UGEP53, UGEP84, UGEP27, UGEP98, UGEP95, UGEP64, UGEP33, UGEP67, UGEP106, UGEP110, UGEP57, UGEP96, UGEP66, UGEP46, UGEP79, UGEP20, UGEP12, UGEP73 and UGEP5 and was since used to assess East African finger millet genetic diversity in two separate studies. <![CDATA[<strong>DNA from tissues of young mice is optimal for genotyping</strong>]]> Background Genotyping of mice is a common procedure in animal facilities. The aim of this study was to compare the quantity and quality of DNA extracted from samples obtained from young mice (YM; 10 d old) and adult mice (AM; 12 weeks old). We collected samples from the tail and ear of YM and AM. We also sampled blood, check cells (via buccal swabs), hair and fecal pellets of AM, and biopsied distal phalanx of YM. We isolated DNA using commercial kits and determined concentrations and purity by spectrophotometry. The integrity of DNA was evaluated by agarose-gel electrophoresis and polymerase chain reaction (PCR). Results DNA in all samples was amplified successfully but the intensities of bands after electrophoresis was heterogeneous. In general, tissues from YM yielded more DNA than those from AM, with differences being statistically significant for ear samples (38 ± 12 ng/μL for YM; 7 ± 3 ng/μL for AM; P = 0.006). In YM, the most DNA was obtained from ear and tail samples, with differences from the amounts obtained from phalanx samples being statistically significant (P = 0.02 and P = 0.005, respectively). In AM, the most DNA was obtained from tail and blood samples. Samples obtained by non-invasive sampling methods in adults resulted in a deficient DNA extraction. Conclusions The results of the present study do not support the previous recommendations for using non-invasive methods to genotype adult animals. The use of newborn tissue samples showed the highest efficiency for DNA extraction. <![CDATA[<strong>Extraction optimization of total triterpenoids from </strong><em><b>Jatropha curcas</b></em><strong> leaves using response surface methodology and evaluations of their antimicrobial and antioxidant capacities</strong>]]> Background Triterpenoids are multifunctional secondary metabolites in plants. But little information is available concerning the actual yield, optimal extraction method and pharmacologic activity with regard to triterpenoids from Jatropha curcas leaves (TJL). Hence, response surface methodology (RSM) was used to optimize the extraction parameters. The effects of three independent variables, namely liquid-to-solid ratio, ethanol concentration and extraction time on TJL yield were investigated. TJL obtained by silica column chromatography was tested against bacterial and fungal species relevant to oral disease and wounds through broth microdilution. Antioxidant activity was assessed using the 2,2-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays. Results A second order polynomial model produced a satisfactory fitting of the experimental data with regard to TJL yield (R2 = 0.983, P < 0.01). The optimum extraction conditions were 16 mL/g (liquid-to-solid ratio), 70% (ethanol concentration) and 50 min (extraction time). Predicted values agreed well with the experimental values. TJL had extraordinarily strong antibacterial and antifungal activities (24.42 µg/mL < MIC < 195.31 µg/mL) against all the tested human pathogens except Bacteroides vulgatus (390.62 µg/mL) and Bacteroides stercoris (781.25 µg/mL). The DPPH and ABTS assays revealed a moderate antioxidant activity of TJL compared with ascorbic acid. Conclusion These results provided reliable scientific basis for further investigation of triterpenoids from J. curcas. <![CDATA[<strong>Efficiency of improved RAPD and ISSR markers in assessing genetic diversity and relationships in </strong><em><b>Angelica sinensis</b></em><strong> (Oliv.) Diels varieties of China</strong>]]> Background Angelica sinensis is a well-known traditional Chinese medicinal plant. We aimed to assess the genetic diversity and relationships in A. sinensis cultivars collected from different locations of China and also some other Angelica species. Results We employed an improved random amplified polymorphic DNA (RAPD) technique for the amplification of DNA materials from ten Angelica cultivars, and the results were verified by inter-simple sequence repeat (ISSR) analysis. Twenty six RAPD primers were used for RAPD, and the amplified bands were found highly polymorphic (96%). Each primer amplified 8-14 bands with an average of 10.25. The cluster dendrogram showed that the similarity coefficients ranged from 0.41 to 0.92. The similarity coefficients were higher among different cultivars of A. sinensis, and lower among different species. Twenty ISSR primers were used for the amplification, and each primer generated 6-10 bands with an average of 7.2 bands per primer. The cluster dendrogram showed that the similarity coefficients ranged from 0.35 to 0.89. Conclusions This study genetically characterized the Angelica species, which might have a significant contribution to the genetic and ecological conservation of this important medicinal plant. Also, this study indicates that the improved RAPD and ISSR analyses are important and potent molecular tools for the study of genetic diversity and authentication of organisms. <![CDATA[<strong>Cloning, expression and characterization of the endoglucanase gene from </strong><em><b>Bacillus subtilis</b></em><strong> UMC7 isolated from the gut of the indigenous termite </strong><em><b>Macrotermes malaccensis</b></em><strong> in </strong><em><b>Escherichia coli</b></em>]]> Background Bacillus subtilis UMC7 isolated from the gut of termite Macrotermes malaccensis has the ability to secrete a significant amount of extracellular endoglucanase, with an enzyme activity of 0.12 ± 0.01 μmol/min/mL. However, for economically viable industrial applications, the enzyme needs to be expressed in a heterologous host to overcome the low enzyme production from the wild-type strain. Results The endoglucanase gene from B. subtilis UMC7 was successfully cloned and expressed. A higher enzyme activity was observed in the intracellular fraction of the recombinant clone (0.51 ± 0.02 μmol/min/mL) compared with the cell-bound fraction (0.37 ± 0.02 μmol/min/mL) and the extracellular fraction (0.33 ± 0.01 μmol/min/mL). The recombinant endoglucanase was approximately 56 kDa, with optimal enzyme activity at 60°C and pH 6.0. The activity of the enzyme was enhanced by the addition of Ca2 +. However, the enzyme was inhibited by other metal ions in the following order: Fe3 + > Ni2 + > Cu2 + > Mn2 + = Zn2 + > Mg2 + > Cd2 + > Cr2 +. The enzyme was able to hydrolyze both low- and high-viscosity carboxymethyl-cellulose (CMC), avicel, cotton linter, filter paper and avicel but not starch, xylan, chitin, pectin and p-nitrophenyl α-d-glucopyranoside. Conclusions The recombinant endoglucanase showed a threefold increase in extracellular enzyme activity compared with the wild-type strain. This result revealed the potential of endoglucanase expression in E. coli, which can be induced for the overexpression of the enzyme. The enzyme has a broad range of activity with high specificity toward cellulose. <![CDATA[<strong>Cellulase production by thermophilic </strong><em><b>Bacillus</b></em><strong> sp</strong>: <strong>SMIA-2 and its detergent compatibility</strong>]]> Background This paper reports the production of cellulase by thermophilic Bacillus sp. SMIA-2 using sugarcane bagasse and corn steep liquor as substrates. Some biochemical properties of the enzyme were also assessed for the purposes of exploiting its potential in the detergent industry, as well as other suitable applications. Results Bacillus sp. produced cellulases when cultivated at 50°C in liquid cultures containing sugarcane bagasse and corn steep liquor. Maximum avicelase (0.83 U mL-1) and CMCase (0.29 U mL-1) activities were reached in 120 h and 168 h of culturing time, respectively. The avicelase and CMCase presented an optimum activity at pH of 7.5 and 8.0, respectively. The maximum stability of avicelase and CMCase was observed at a pH range between 6.5-8.0 and 7.0-9.0 respectively, where they retained more than 70% of their maximum activities after incubation at room temperature for 3 h. The optimum temperature of avicelase and CMCase was 70°C, and both enzymes remained 100% stable until the treatment at 60°C for 1 h. Bacillus sp. cultures also released proteases into the culture medium, but the cellulases were resistant to protease digestion. The compatibility of cellulases varied with each laundry detergent tested, being more stable in the presence of Ultra Biz® and less with Ariel®. In addition, the enzyme was stable in sodium dodecyl sulfate and RENEX-95, and was inhibited by TritonX-100 and H2O2. Conclusions The properties presented by Bacillus sp. SMIA-2 suggest that this organism might become a potential source of lignocellulose-degrading enzymes for industrial applications such as in the detergent industry. <![CDATA[<strong>The power of 28 microsatellite markers for parentage testing in sheep</strong>]]> Background In sheep breeding, there are situations where relationships recorded at the farm among pedigrees such as parent-offspring, full-sibs or half-sibs need to be tested. A panel of 28 microsatellite (MST) markers was tested to provide accurate pedigree information and resolve the common problem of significant error in pedigree records in Merino sheep. Three different flocks of Australian Merino sheep were investigated. A private farm flock represents a flock with no record availability. Two other flocks were maintained under good managements of full keeping records and being selected for high and low parasite resistances. Results In the studied panel, eight MSTs provided an average of Polymorphic Information content (PIC) equal to 0.65 or more in order to be sufficient to make an accurate and successful DNA-based parentage analysis. The panel of twenty-eight MST loci was obviously sufficient for providing 100% accurate pedigree and genotyping data. DNA-based pedigree records were constructed and all significant pedigree record errors were eliminated. Conclusions These results were used for further study of population genetic parameters such as recombination and haplotyping which heavily based on pedigree information. Nevertheless MST based parentage testing is still available and affordable in most countries and for each farmer with reasonable cost in comparison with fast growing SNP based parentage technologies. <![CDATA[<strong>PPAR</strong><strong>γ</strong><strong>, FAS, HSL mRNA and protein expression during Tan sheep fat-tail development</strong>]]> Background The objective of this study was to investigate proliferator-activated receptor (PPARγ), fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) mRNA and protein expression in fat tails of Tan sheep. Rams from different developmental stages (aged 3, 6, 9, 12, 15 and 18 months) were selected, and their tail measurements including length (L), width (W) and girth (G) were recorded. The mRNA and protein expressions of PPARγ, FAS and HSL were examined by quantitative real-time polymerase chain reaction (PCR) and Western blot. Results The tail measurements increased with age. We observed no significant differences (P &gt; 0.05) of PPARγ mRNA expression between ages 9 and 15 months, and between 12 and 15 months; FAS mRNA expression levels at each developmental stage were observed significantly in Tan sheep (P < 0.05); HSL mRNA expression with no significant differences were only observed between 6 and 15 months (P &gt; 0.05). Significant differences (P < 0.05) of PPARγ, FAS and HSL protein expressions at each developmental stage were observed in Tan sheep. Conclusion We observed that the mRNA expression patterns of PPARγ and FAS decreased first before they increased again and then this process repeated. Conversely, the mRNA expression patterns of HSL increased first before they decreased and then this process repeated. The protein expression patterns of PPARγ and FAS decreased first before they increased again and then this process repeated. Conversely, the protein expression pattern of HSL increased first before it decreased again and then increased again. <![CDATA[<strong>The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of </strong><em><b>Clostridium butyricum</b></em><strong> DSP1</strong>]]> Background The production of biofuels from renewable energy sources is one of the most important issues in biotechnology today. The process is known to generate various by-products, for example glycerol that is obtained in the making of biodiesel from rapeseed oil. Crude glycerol may be utilized in many ways, including microbial conversion to 1,3-propanediol. The main drawback of that technology is the use of high concentrations of glycerol, which inhibits the growth of bacterial cells. Results This study investigated the impact of crude glycerol on Clostridium butyricum DSP1 and its ability to adapt to an environment of high osmotic pressure. It was found that a crude glycerol concentration of up to 70 g/L did not have an inhibitory effect on C. butyricum DSP1. Adaptation procedures involving the passage of metabolically active biomass from a fermentation medium with a lower concentration of crude glycerol to one with a greater substrate concentration allowed breaking the barrier of high osmotic pressure (150 g/L crude glycerol) and receiving a 1,3-PD concentration of 74 g/L in a batch culture operation. The work looked into intracellular modifications shown by proteomic profiling in order to explain the mechanisms underlying the response and adaptation of bacterial cells exposed to unfavorable environmental conditions. Conclusions This study of the effect of glycerol on the growth and metabolism of C. butyricum DSP1 demonstrated that the maximum substrate concentrations that do not inhibit the metabolic activity of bacterial cells are 90 g/L and 70 g/L for pure and crude glycerol, respectively. <![CDATA[<strong>Total RNA quality of lyophilized and cryopreserved dormant grapevine buds</strong>]]> Background Plant tissues must be preserved in their collection state, especially for genome-wide expression profile studies. Lyophilization is a feasible, affordable tool when fresh tissues cannot be shipped at ultralow temperatures from their origin to the place of analysis. In this study, the total RNA quality of dormant grapevine buds (Vitis vinifera L. cv. ‘Flame Seedless’) of freeze-dried samples stored at room temperature conditions was evaluated and compared to that of cryopreserved (-80°C) grapevine buds. Results Good yield and quality of RNA were obtained from freeze-dried dormant buds stored at room temperature for 0, 3 and 6 weeks after they were lyophilized. Further experiments confirmed that the extracted total RNA could be used for actin and β-tubulin PCR gene amplification. Conclusion High-quality RNA that is useful for downstream applications was obtained from freeze-dried dormant grapevine bud tissue, similarly to the RNA obtained from cryopreserved dormant grapevine buds. <![CDATA[<strong>Engineering </strong><em><b>Escherichia coli</b></em><strong> for autoinducible production of n-butanol</strong>]]> Background Escherichia coli does not produce n-butanol naturally, but can be butanologenic when related enzymes were expressed using inducible elements on plasmids. In this study we attempted to confer E. coli strain capability of automatic excretion of the chemical by employing a native anaerobic promoter. Also, a novel DNA kit was designed for PCR preparation of linear DNA fragments to perform strain modification. The kit is primarily composed of two mother vectors, co-transformation of linear DNAs into E. coli can simultaneously introduce two butanol synthetic operons into the chromosome and create two in-frame gene deletions at targeted native loci. Results E. coli strain Bw2V carries plasmid pCNA-PHC and pENA-TA, both utilizes native anaerobic promoter Phya for the expression of butanol synthetic enzymes. When Bw2V was subjected in anaerobic fermentation using medium containing extra glucose, the accumulated n-butanol in the broth was up to 2.8 g/L in bioreactor; as the genetic element expressing the same pathway was introduced into the genome, the titer of butanol was 1.4 g/L. Conclusions The expression system using Phya is effective in applications that involve expression plasmids as also applicable in ectopic expression as single copy on the chromosome. Results imply that Phya can be subjected for broader application in bioproduction of more feedstock chemicals.