بررسی کارآیی چهار روش استخراج DNA از زنبورعسل کوچک Apis florea (Hymenoptera: Apidae) جهت انجام آزمایشات ژنتیکی

نوع مقاله: علمی پژوهشی-فارسی


دانشجوی سابق کارشناسی ارشد دانشگاه کردستان


استخراج DNA اولین مرحله در انجام آزمایشات ژنتیکی است، لذا کیفیت و کمیت مناسب DNA استخراج شده در مطالعات مولکولی امری بسیار ضروری می باشد. از این رو دستیابی به روش یا روش ­هایی که به سهولت استخراج DNA از زنبورعسل کوچک Apis florea Fabricius  که یکی از دو گونه زنبورعسل موجود در ایران است کمک کند، می ­تواند حائز اهمیت باشد. این گونه زنبورعسل به دلیل پراکندگی گسترده و همچنین ایفای نقش مستقیم در گرده افشانی گونه‌های زراعی، باغی و جنگلی از اهمیت مطالعاتی زیادی برخوردار است.  به همین دلیل در این مطالعه چهار روش استخراج DNA شامل بهینه نمکی، فنل-کلروفرم، CTAB و CTAB+SDS به منظور گزینش مناسب­ ترین روش استخراج DNA زنبورعسل کوچک مورد ارزیابی و آزمایش قرار گرفت. سپس کیفیت و کمیت DNA استخراجی با استفاده از روش­ های اسپکتوفتومتری و ژل آگارز مورد مقایسه قرار گرفت. با توجه به کمیت و کیفیت DNA استخراج شده، روش CTAB+SDS برای استخراج DNA از زنبورعسل کوچک قابل توصیه است.


عنوان مقاله [English]

Evaluation of four different DNA extraction methods from dwarf honeybee Apis florea (Hymenoptera: Apidae) for genetic experiments

نویسندگان [English]

  • D. Najafzadeh
  • S. Lalegani
Former M.Sc. student, University of Kurdistan
چکیده [English]

Background and Objectives
DNA extraction is the first step of genetic experiments. In this regard, quality and quantity of extracted DNA plays an important role in molecular studies. This experiment was conducted in order to achieve a novel method that helps us to extract DNA from Apis florea F (one of two important Iranian honeybee). This species is regarded as an important honeybee due to its world-wide distribution and direct role in pollination of field crops, orchards and forests.
Materials and Methods
Samples were collected randomly from the hives of dwarf honeybee in Khuzestan province. In the current research, DNA was extracted from the thorax tissues of worker bees. Totally, four DNA extraction methods were evaluated including the optimal salting out, phenol-chloroform, CTAB and CTAB+SDS. To evaluate the quality of extracted DNA, spectrophotometry, electrophoresis and agarose gel methods were applied to compare quantity and quality of DNA extraction.
Quantity and quality of extracted DNA was evaluated by spectrophotometry based on the absorption ratio 260/280 and electrophoresis on agarose gel. Based on the absorption ratio, for the methods of salting out, phenol-chloroform, CTAB and CTAB + SDS were 1.15 ± 0.01, 1.37 ± 0.01, 1.72 ± 0.02 and 1.82 ± 0.01, respectively. Duncan multiple range test revealed that CTAB+SDS was significantly different from other methods for evaluation of DNA extraction.
Based on the results, the quality of DNA visible bands on the agarose gel can be considered as the option to evaluate the quality of DNA. On the other hand, twice washing in the CTAB + SDS method reduced protein contamination significantly. Furthermore, it could be mentioned that all the DNA extraction methods used in this study seems to be suitable in polymerase chain reaction test, but CTAB+SDS resulted in higher DNA quality and quantity. We referred to this thorax tissue as “thoracic muscle mass”, which could be as candidate tissue to obtain larger quantities of DNA.

کلیدواژه‌ها [English]

  • Apis
  • CTAB
  • DNA
  • Extract
  • Genetic
Arias, M.C., and Sheppard, W.S. 2005. Phylogenetic relationships of honeybees (Hymenoptera: Apinae: Apini) inferred from nuclear and mitochondrial DNA sequence data.Molecular Phylogenetic and Evolution, 37 (1): 25-35.‏
Berthomieu, P., and Meyer, C. 1991. Direct amplification of plant genomic DNA from leaf and root pieces using PCR,” Plant Molecular Biology, 17 (3): 555-557.
Delarua, P., Galian, J., Serrano, J. and Moritz, R.F.A. 2001. Genetic structure and distinctiveness of Apis mellifera L. populations from the Canary Islands. Molecular Ecology, 10: 1733-1742.
Doyle, J.J., and Doyle, L.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19: 11-15.
Estoup, A., Garnery, L., Solignac, M., and Cornuet, J. 1995. Microsatellite variation in honey bee (Apis mellifera L.) populations: Hierarchical genetic structure and test of the infinite allele and stepwise mutation models. Genetics, 140: 679-695.
Evans, D.J., Schwarz, R.S., Chen, Y.P., Budge, G., Cornman, R.S., Delarua, P., Miranda, J., Foret, S., Foster, L., Gauthier, L., Genersch, E., Gisder, S., Jarosch, A., Kucharski, R., Lopez, D., Lun, D.M., Moritz, R., Maleszka, R., Muñoz, I. and Pinto, M.A. 2013. Standard methods for molecular research in Apis mellifera. International Bee Research Association. Journal of Apicultural Research, 52 (4):1-53.
Franck, P., Garnery, L., Loiseau, A., Oldroyd, B. P., Hepburn, H.R., Solignac, M., and Cornuet, J.M. 2001. Genetic diversity of the honeybee in Africa: microsatellite and mitochondrial data. Heredity, 86 (4): 420-430.
Gari, M.A., Abuzenadah, A.M., Chaudhary, A.G., Al-Qahtain, M.H., Al- says, F.M., and Dmanhouri, G. 2006.  Pilot study of DNA extraction from archival unstained bone marrow slides: comparison of three rapid methods. African Journal ofBiotechnology, 5(6): 532-535.
Garnery, L., Franck, P., Baudry, E., Vautrin, D., Cornuet, J.M., and Solignac, M. 1998. Genetic diversity of the west European honeybee (Apis mellifera and A. M. iberica). I. Mitochondrial DNA, Genetics Selection Evolution, 30: 531-547.
Hepburn, H.R., and Radloff, S.E. 2011. Honeybees of Asia. Springer-Verlag, Berlin, Heidelberg, P. 669.
Irfan, K., Marina, D.M.,Ayca, O., and Walter, S.S. 2006. Genetic characterization of honeybee (Apis mellifera Cypria) populations in northern Cyprus. Apidologie, 37: 547-555.
Kreuzer, H., and Adrianne, M. 2003. Recombinant DNA and Biotechnology. Mohamadreza Shakibai Publication, Kerman. P. 200. (In Persian).
Kury, F., Schneeberger, C., Sliutz, G., Kubista, E., Salzer, H., Medl, M. and Spona, J. 1990. Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation. Oncogene, 5(9): 1403-1408.
Mossadegh, M. 2014. Know the dwarf honey bee Apis(micrapis)florea F. (hymenoptera: apidae). Pakpendar publocation, Karaj. P. 570. (In Persian with English abstract).
Naraqi, A. 2011. Honeybee and Honeybee keeping, Aeej Publication. Tehran. P. 336. (In Persian).
Nicholl D.S.T. 1960. An introduction to genetic engineering. Cambridge University Press. 1996. 1-30.
Poljak, M., Barlic, J., Seme, Avsic-Zupanc, K., and Zore, T. 1995. Solation of DNA from archival Papanicolaou stained cytological smears using a simple salting-out procedure. Clinical and Molecular Pathology, 48(1): 55-56.
Ruttner, F., Pourasghar, D., Kauhausen. D. 1985. Die Honigbienen des Iran. I. Apis florea Fabricius. Apidologie 16: 119-12.
Ruttner, F. 1988.  Biogeography and taxonomy of honeybees. Springer-Verlag, Berlin, Heidelberg GmbH, P. 285.
Shouhani, H., Dousti, A., Radjabi, R., and Zarei, M. 2012. Application of ISSR molecular marker in the genetic diversity of bee (Apis mellifera L.) population in some parts of Iran. Journal of Bioscience and Bioengineering, 3(2): 127-131.
Strange, J., Garnery, L., and Sheppard, W. 2007. Morphological and molecular characterization of the Landes honeybee (Apis mellifera L.) ecotype for genetic conservation. Insect Conservation, 12: 527-537.
Trevor, B., and Julian, B. 2006. Transcription and structure Gen. (Elahi, E., Emadi, S., Ghorbani Nezami, A.) Sina sharif publication, Tehran. P. 136. (In Persian).
Yogesh , K., and Khan, M. 2014. Genetic variability of European honey bee, Apis mellifera in mid hills, plains and tarai region of India. African Journal of Biotechnology, 13(8): 916-925.
Watson, J. 2004. Transcription and structure Gen. (Samadi, A., and Pasalar, P.) University of Tehran publication. Tehran. P. 436. (In Persian).