Open Access Short Research Article

Use of Gene Specific Universal Primers for Isolation of DNA Sequences Encoding Laccase Enzyme from a Wild Isolate of Schizophyllum commune

Vidya Pradeep Kumar, Atul P. Kolte, Arindam Dhali, Chandrashekar Naik, Manpal Sridhar

Biotechnology Journal International, Page 1-11
DOI: 10.9734/BJI/2017/37022

Laccase enzymes plays a vital role in innumerable biotechnological applications and hence their large scale production has stimulated considerable research. In the present study, degenerate universal primer pairs were employed to isolate laccase gene from a wild isolate of laccase producing white rot fungi Schizophyllum commune. Primer pairs for this fungal isolate were also designed using laccase specific consensus sequences for fungi. The PCR product of 1000 bp amplicon was visualized on agarose gel using the degenerate primer pair Cu1F/Cu3R. Matching of the sequenced gel purified DNA sequence resembled most of the putative phosphatases involved in cell cycle with 100% identity to S. commune. Here we report the false negative results obtained upon use of laccase specific degenerate primers as well as other primers specific for the genus. These failed to contribute towards isolation of laccase gene even after optimization of PCR conditions in terms of reaction volume, annealing temperature, number of cycles, touchdown PCR and gradient PCR. These findings constitute a practical guide for researchers addressing amplification of transcripts of this biotechnologically important enzyme from the not so well characterized genus of Schizophyllum.

Open Access Original Research Article

Molecular Markers and Postulation Study of Leaf Rust Resistance Genes in Various Egyptian Wheat Cultivars

Hanaa Abouzied, Eman El Argawy, Walid El-Orabey

Biotechnology Journal International, Page 1-13
DOI: 10.9734/BJI/2017/36175

The current investigation was carried out at seedling stage under greenhouse condition to: (1) postulate leaf rust resistance genes in ten Egyptian spring wheat cultivars at seedling stage and (2) Verify the presence of resistance genes in the selected cultivars using specific molecular markers linked to leaf rust resistance genes. The ten Egyptian spring wheat cultivars i.e. Misr 1, Misr 2, Sids 12, Sids 13, Gemmeiza 9, Gemmeiza 10, Gemmeiza 11, Sakha 93, Sakha 94 and Giza 168 as well as eight monogenic lines carrying single gene for leaf rust resistance i.e. Lr 21, Lr 25, Lr 32, Lr 35, Lr 37, Lr 39, Lr 47, and Lr 51 were tested at seedling stage using 15 leaf rust pathotypes i.e. BDGGK, CPPTB, DMKRT, FKRNK, FTFNB, KTSQG, LDDLD, LFGLL, NRRLS, NTTSR, PTKLN, STRTQ, TJKPR, TQTMJ and TTJBC. Eight specific molecular markers were known to be linked with the Lr,s resistance genes, i.e. Lr 21, Lr 25, Lr 32, Lr 35, Lr 37, Lr 39, Lr 47, and Lr 51 were used to verify the presence of these genes in the tested Egyptian wheat cultivars. The postulation test theorized the presence of Lr 25, Lr 32, Lr 35, Lr 37 and Lr 47 in Sids 12. Moreover, the wheat cultivar Misr 1 may have four genes i.e. Lr 25, Lr 35, Lr 39 and Lr 51. The wheat cultivars Misr 2, Sids 13, Sakha 94 and Giza 168 may have two genes i.e. Lr 35 and Lr 39 in the wheat cultivar Misr 2 and the same two genes Lr 35 and Lr 37 in the three wheat cultivars Sids 13, Sakha 94 and Giza 168. On the other hand, the wheat cultivars Gemmeiza 9 and Sakha 93 may have only one gene i.e. Lr 35 and Lr 32, respectively. While, the wheat cultivars Gemmeiza 10 and Gemmeiza 11 did not have any of the tested Lr genes under study using the fifteen tested pathotypes of P. triticina but it may be carrying some other genes. In the same way, all of the tested wheat genotypes may also carries additional resistant gene (s).The molecular markers survey with specific markers revealed that, the specific markers for Lr 21 and Lr 47 were detected in all tested cultivars. The marker for Lr 32 was recognized in four cultivars i.e. Gemmeiza10, Sakha 93 Sakha 94 and Giza168. The marker for Lr 39 was identified in all cultivars except Sakha 94. The markers for Lr 25, Lr 35, Lr 37 and Lr 51 were not found in any the tested cultivars. This study may add valuable information about resistance genes in the Egyptian wheat cultivars which may be used in pyramiding more genes in each cultivar or used as donor parents in different wheat genetic improving programs.

Open Access Original Research Article

Potentialities of Yeast Strains to be Used as Freeze-Drying Starters for the Production of Traditional Sorghum Beer in Côte d’Ivoire

Wahauwouélé Hermann Coulibaly, Fatoumata Camara, Djegba Marie Toka, Koffi Maïzan Jean-Paul Bouatenin, Alfred K. Kouamé, Youan Charles Tra Bi, Anne Abot, Koffi Marcellin Djè

Biotechnology Journal International, Page 1-15
DOI: 10.9734/BJI/2017/36851

Freeze-drying is a well-known dehydration method widely used to preserve microorganisms. In order to produce freeze-dried yeast starter culture for the brewing purpose of African traditional sorghum beer, we tested tolerance to ethanol stress and evaluated the relative expression level of genes TIF 11 and YJL144W encoding the hydrophilins. Among the strains tested, the best viability rate to ethanol stress (7.5% ethanol (v/v)) was found with Saccharomyces cerevisiae   F12–7 and   Candida tropicalis   C0–7 respectively with 95% and 80%. For Saccharomyces cerevisiae strains, the strain F12-7, which had distinguished itself from other strains in previous tests, the TIF11 and YJL144W genes were the least expressed. For C. tropicalis strains, the statistical analyzes of the relative expression levels from the Tukey test revealed no difference between the strains for the 2 genes (P> 0.05).

Open Access Original Research Article

Prokaryotic Diversity of the Alkaline Lake Acıgöl, Turkey by Using Culture-dependent and Culture-Independent Methods

Gamze Başbülbül, Haci Halil Biyik, Erman Oryaşın, Bülent Bozdoğan

Biotechnology Journal International, Page 1-16
DOI: 10.9734/BJI/2017/36909

Aims: The prokaryotic diversity of Acıgöl lake, Turkey, was studied in samples taken from soil, water and mud. Culture -dependent and -independent methods were used to analyse the results.

Methodology: Bacterial cultures were isolated by using six different growth media. Isolates were identified according to their 16S rDNA sequence analysis. Liquid media were used to test antibacterial substance production of the isolates. Bacillus cereus, Serratia marcescens, Pectobacterium carotovorum, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, Micrococcus luteus, Escherichia coli and Listeria innocua were used as indicator bacteria. The extracted DNA samples taken from soil, water and mud were used in PCR reactions to amplify 16S rDNA genes by using universal bacterial primers. Amplicons were cloned by TA cloning after which white colonies were selected and colony PCR were applied for the amplification of the fragments. Each amplicon obtained from clones was sequenced and similarities were compared to those of the other bacterial groups. The DNA extracted from water sample was also used to amplify archeal 16S rDNAs. For the amplification reaction, the primers used were specific to the archaeal domain and their amplicons were cloned by TA cloning. Randomly picked white colonies were used for M13 PCR. The sequences of amplicons were analyzed by BLAST to determine the highest similarities with the other archeal groups.

Results: In this study, fourty-nine strains were identified as belonging to Bacillus, Halomonas Enterococcus, Exiquobacterium, Piscibacillus, Haloalkalibacillus, Aerococcus, Acinetobacter, Lysinibacillus, Oceanobacillus, Planococcus, Micrococcus genera according to 16S rDNA analysis. Among these isolates, the GBA17, GBA34 and GBA36 weakly inhibit the growth of M. luteus. The analysis of 16S rDNA from different environmental samples suggests that the bacterial clones belong to the class of Gemmatimonadetes (n=1), Alphaproteobacteria (n=4), Flavobacteria (n=1), Opitutae (n=2), Gammaproteobacteria (n=1), Saprospira (n=1), Verrucomicrobia (n=1). All of the identified archeal clones were affiliated to the Class Halobacteria with 12 members belonging to the Halobacteriales order and two members belonging to the Haloferaceles order. No match was found between the taxa determined by culture-dependent method and those determined by independent method. Growth media used in the isolation experiments might not support the growth of extreme halo-alkaliphilic bacteria found as a result of cloning.

Haloalkaliphilic and alkaliphilic isolates obtained in our study may be ideal candidates as industrially important substance producers.

Open Access Original Research Article

Comparative Analysis of DNA Extraction Methods in Two Popular Varieties of Finger Millet (Eleusine coracana) from Ethiopia

Aklilu Banjaw Leza, Sunil Tulshiram Hajare, Nitin M. Chauhan

Biotechnology Journal International, Page 1-7
DOI: 10.9734/BJI/2017/33145

Aims: The aim of this investigation was to standardize the genomic DNA isolation protocol to obtain high quality and quantity DNA for genomic analysis. To accomplish this task, dry leaves of finger millet which are rich in polysaccharides and polyphenols and seeds were utilized as study group.

Methodology: Two popular finger millet varieties of Ethiopia (Tadesse and Degu) were selected and obtained from Hawassa Agricultural Research Institute, Hawassa, Ethiopia. DNA isolation was carried out by two most popular and reliable methods i.e. Cetyl Trimethyl Ammonium Bromide (CTAB) and Dodecyl Trimethyl Ammonium Bromide (DTAB) method with little modifications. The quality of DNA obtained through the two methods was comparatively evaluated.

Results: The CTAB method proved its superiority over DTAB. The purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.76 to 1.78, which also suggested that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds. However, the DTAB method failed to extract sufficient quantity and quality of genomic DNA for further genomic analysis.

Conclusions: Based on our study, our protocol can be useful for other difficult cereal crops in the future.