Isolation and Molecular Identification of Some Blue-Green Algae (Cyanobacteria) from Freshwater Sites in Tokat Province of Turkey
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Turkish Journal of Agriculture - Food Science and Technology, 5(11): 1371-1378, 2017 Turkish Journal of Agriculture - Food Science and Technology Available online, ISSN: 2148-127X www.agrifoodscience.com, Turkish Science and Technology Isolation and Molecular Identification of Some Blue-Green Algae (Cyanobacteria) from Freshwater Sites in Tokat Province of Turkey Tunay Karan1*, Ramazan Erenler2, Zekeriya Altuner3 1Division of General Biology, Department of Biology, Faculty of Arts & Sciences, Gaziosmanpasa University, 60250 Tokat, Turkey 2Division of Biochemistry, Department of Chemistry, Faculty of Arts & Sciences, Gaziosmanpasa University, 60250 Tokat, Turkey 3Department of Biology, Faculty of Science, Gaziosmanpasa University, 60250 Tokat, Turkey A R T I C L E I N F O A B S T R A C T Collected blue-green algae (cyanobacteria) from freshwater sites throughout Tokat Research Article province and its outlying areas were isolated in laboratory environment and their morphological systematics were determined and also their species identifications were Received 15 August 2017 studied by molecular methods. Seven different species of blue-green algae collected from Accepted 20 September 2017 seven different sites were isolated by purifying in cultures in laboratory environment. DNA extractions were made from isolated cells and extracted DNAs were amplified by Keywords: using PCR. Cyanobacteria specific primers were used to amplify 16S rRNA and Algae phycocyanine gene regions using PCR. Phylogenetic identification of species were 16S rRNA conducted by evaluation of obtained sequence analysis data by using computer software. Cyanobacteria According to species identification by sequence analysis, it was seen that molecular data Molecular identification supports morphological systematics. Phycocyanine *Corresponding Author: E-mail: [email protected] DOI: https://doi.org/10.24925/turjaf.v5i11.1371-1378.1470 Introduction Cyanoprokaryotes are unique among microorganisms Tallus fiber and only present in cyanobacteria (Sukatar, due to having chlorophyll a and their ability to make 2002; Altuner 2010). Morphological traits are taken as photosynthesis (Singh et al., 2011). They have gram basis for classification of cyanobacteria. However, negative cell walls and their peptidoglycan layers are morphological traits might be altered due to changes in ranging in 10–200 nm of thickness (Gademan and environmental and developmental conditions. Strain Portman, 2008). Dominant pigments present in diversity in cultures can be inhibited by using elective cyanobacteria are chlorophyll a, chlorophyll c and culture conditions. These reasons are making the phycocyanine which contributing to their distinct blue- development of molecular techniques for identification of green colorization. Additionally they have different kinds cyanobacteria a necessity (Lyra et al., 2001). of color pigments such as β-carotene and xanthophyll. Most frequently used marker gene is 16S rRNA gene Also some cyanobacteria species contains phycoerythrin for both identification of microorganisms and evaluation pigments which is one of phycobilin bilanes and of relationships between them. While 16S rRNA gene attributed to red colorization (Altuner, 2010). contains many evolutionarily conserved sequences, many Cyanobacteria can either be unicellular or aggregates species specific variable sequences are also present in 16S of many cells forming a colony. Those of unicellular rRNA gene. Species level identification is possible by cyanobacteria mostly assume forms of bacilli, discoidal, amplifying these variable sequences using PCR. Precise actinoidal or fibrillary shapes whereas colony forming identifications of species of Geitlerinema strain PCC cyanobacteria mostly assume forms of spherical shapes 9452 (Microcoleus sp. strain 10 MFX) and Oscillatoria (Whitton and Potts, 2000). Certain cyanobacteria species limnetica have been shown by amplification of 16S rRNA contain special cells called as heterocyst which are genes (Boyer et al., 2001). Cylindrospermum Kutz. Ex. smaller than akinete and responsible for fixating free Bornet et Flahault differentiated from Nostocacea family nitrogen of air. These cells are among cells forming due to their terminal heterocysts and paraheterocystic Karan et al., / Turkish Journal of Agriculture - Food Science and Technology, 5(11): 1371-1378, 2017 akinetes and 45 taxa of this genus identified (Guiry and Irvine, CA; USA) in accordance with manufacturer Guiry, 2014; Johansen et al., 2014). Even though less instructions. Amount and purity of extracted DNA were frequently used, some researchers are using protein quality checked by using nanodrop spectrophotometry coding gene regions of rpoB, rpoC1, recA, rpoD1 and (DeNovix, Wilmington, DE; USA) at 260–280 nm nifH as alternative templates for phylogenetic analysis of wavelength absorbance. cyanobacteria (Saker et al, 2005; Premanandh et al, 2006). In another study, strains of Synechococcus have PCR Procedures been determined by phylogeny using phycocyanine gene Obtained genomic DNAs were first amplified by sequences (Robertson et al, 2001). In this study it is aimed using universal bacterial primers for 16s gene region for to isolate, purify and molecularly identify various all the cyanobacteria samples. Universal bacterial primer cyanobacteria collected from different freshwater sites on that were used have sequences for forward primer as 27F: riverbank of Yesilirmak in Tokat Province of Turkey. 5’-AGA GTT TGA TCC TGG CTC AG-3’ and for reverse primer as 1492R: 5’-TAC GCG CTA CCT TGT Material and Methods TAC GAC-3’. Afterwards, 16s gene region PCR amplification, phycocyanine gene region was conducted Sample collection by using forward primer of PCβF: 5’-GGC TGC TTG Seven different stations were chosen on pelagic sites TTT ACG CGA CA-3’ and reverse primer of PCαR: 5’- of Yesilirmak riverbank throughout Tokat province of CCA GTA CCA CCA GCA ACT AA-3’ just for Turkey and its outlying freshwater branches. Coordinates Anabaena oryzae and Nostoc linckia taxon. Roche of chosen sites were determined by using geographical FastStartTaq DNA Polymerase kit (Roche Life Sciences, positioning system (GPS) and presented in Table 1. Istanbul, Turkey) was used for PCR reactions. PCR Collected freshwater samples were transferred into 1 L mixtures were prepared in 0.2 ml sterile PCR tubes by plastic canisters and were delivered to microalgae additions of following reagents: 2.5 µl 2 mM 10x PCR culturing laboratory. buffer; 0.75 µl 10 mM dNTP; 2.5 µl 0.2 µM 10x primers (separately for each primer); 0.25 µl 2 U Taq DNA Cyanobacteria Isolation Polymerase; 100 ng genomic DNA. Total mixture volume Delivered freshwater samples were first tromped by add up to 25 µl by filling with ddH O. DNA filter paper (GF/C Filter, Whatman, GE Life Sciences, 2 amplifications were done using thermal cycler (Bio-Rad PA; USA) and transferred into petri dishes. Filtrated Turkey, Istanbul, Turkey) in following configuration for samples were sterilized under UV light for 10 min in a 30 cycles: Initial denaturation at 95°C for 4 min; sterilization chamber. Samples in petri dishes were Denaturation at 95°C for 55 sec; Annealing at 60°C for 45 incubated in enrichment solution (F/2 solution) until the sec; Extension at 72°C for a minute. At the last step of identification stage and dishes were kept in acclimation amplification, a final extension at 72°C for 7 min was chamber (Sanyo MLR 351, Sanyo, Tokyo, Japan) at 26°C applied. PCR products were subjected to electrophoresis under 2465 lux illumination with 12:12 hours light/dark inside 1.5% agarose gel by applying 80 V current for 45 cycle (Andersen, 2005; Teneva et al, 2012). min. Then, stained by ethidium bromide and screened Morphological traits were examined under light under UV transilluminator cabin (Saker et al, 2005; microscope (Olympus CX31-P, Olympus, Japan). Species Premanandh et al, 2006; Green et al, 2008). going to be isolated were determined and mechanically isolated under inverted microscope by using micropipette Sequencing and Phylogeny and microinjection (Rai and Rajashekhar, 2016). The PCR products of 16s rRNA and phycocyanin Following the mechanical isolation, strains were gene regions were sequenced and edited at REFGEN inoculated into 1.5% agar and appropriate nutritional (Gene Research and Biotechnology Ltd. Sti., Ankara, media (Bristol BG11 and BG11°, HiMedia Labs, TURKEY). Additional sequences (nineteen phycocyanin Mumbai, India) by using line inoculation method. gene and seventeen 16s rRNA gene sequences, Table 2) Procedures were repeated until only one strain remain for further phylogenetic analyses were acquired from isolated and pured (Galhanoa et al, 2011). GenBank (https://www.ncbi.nlm.nih.gov/genbank/). All of 16s rRNA and phycocyanin genes sequences Morphological Characterization were aligned using the Clustal-W algorithm in Morphological differentiations based on either strains GENEIOUS version 10.1.3 (http://www.geneious.com, were filamentous or unicellular and if filamentous strains Kearse et al., 2012). contained heterocysts were conducted first. Widths and Limnothrix redekei (HE974998.1) and Cyanothece sp. heights of examined strains were measured by using (CP002198.1) were used as outgroups in analyses. micrometer apparatus in microscope ocular and Phylogenetic trees were constructed using the maximum photographs were taken. Systematics of purified strains likelihood (ML) algorithm presented in RAxML plugin were done in accordance to algae database (accessed