تجزيه و تحليل بيوانفورماتيكي و فيلوژنتيكي ژن COX3 ميتوكندري شترهاي تك كوهانه و دوكوهانه ايران

Bioinformatics and Phylogenetic Analysis of Mitochondrial COX3 Gene in Iranian Camelus Dromedaries and Camelus Bactrianus

نژادهای بومی ایران بخشی از سرمایه ملی تلقی، بنابراین حفظ آن¬ها اهمیت ویژه¬ای دارد. توجه بیشتر به این نژادها از دیدگاه ژنتیك حفاظتی با توجه به كاهش شدید جمعیت آن¬ها در برخی مناطق، از اهمیت زیادی برخوردار است. هدف از انجام این مطالعه، بررسی بیوانفورماتیكی و فیلوژنتیكی توالی نوكلئوتیدی زیرواحد 3 كمپلكس سیتوكروم اكسیداز (COX3) میتوكندری در شترهای تك كوهانه و دوكوهانه ایرانی بود. برای این منظور 10 نمونه خون از هر یك از این دو گونه جمع آوری شد (20 نمونه). پس از استخراج DNA، قطعه 979 جفت بازی از ژنوم میتوكندریایی با استفاده از واكنش زنجیره‌ای پلیمراز تكثیر گردید. توالی‌یابی با روش اتوماتیك سانگر صورت گرفت و سپس توالی¬های بدست آمده با توالی‌های حاصل از مطالعات دیگر، مقایسه شدند. نتایج به دست آمده نشان داد كه هیچ تفاوت نوكلئوتیدی بین توالی COX3 نمونه¬های مختلف تك كوهانه و نمونه¬های مختلف دوكوهانه ایرانی وجود نداشته و همچنین توالی¬های نوكلئوتیدی در این دو گونه به ترتیب با شتر تك كوهانه (شتر عربی) با شماره دسترسی NC_009849 و شتر دوكوهانه (شتر باختری) با شماره دسترسی NC_009628، همولوژی 100 درصد دارند. مقایسه توالی¬های نوكلئوتیدی و اسیدآمینه¬ای و همچنین ساختار سه بعدی COX3 در گونه¬های شتر ایرانی نشان داد كه این دو گونه دارای فاصله ژنتیكی نزدیكی هستند. تجزیه و تحلیل فیلوژنتیكی مشخص كرد كه این دو گونه در میان خانواده شترسانان، با Lama guanicoe كمترین قرابت را دارند.

Introduction Camels belong to the family of Camelidae, suborder of Tylopoda, order of artiodactyla and class of mammalians. The family Camelidae has two old world species, double-humped camel (CAMELUS BACTRIANUS) and single-humped camel (CAMELUS DROMEDARIES) and four new world (tribe Lamini) species, guanaco (LAMA GUANICOE), llama (LAMA GLAMA), alpaca (LAMA PACOS) and vicuna (LAMA VICUGNA or VICUGNA VICUGNA) at present time. The single-humped camel inhabits Afro-Arabia, Ethiopia and west Central Asia while the double-humped inhabits eastern Central Asia and China). Camel has been historically and economically an important species worldwide especially in the Africa and Asia. Camel has unique characteristics enable it to adapt its desert environment. The total worldwide camel population at present estimated to be about 23 million in the world. Somalia and Sudan together hold approximately 50% of the whole camel population. In the last 40 years, the number of camels has increased by almost 45%. Iranian native species are considered as part of the national capital so their preservation is so important. Due to severe decrease in their population in some areas, more attention to conservation genetics perspective of these species is very important. The aim of this study was to bioinformatics and phylogenetic analysis of mitochondrial sequence of cytochrome c oxidase subunit 3 (COX3) in Iranian Camelus dromedaries and Camelus bactrianus. Materials and Methods For this purpose 10 blood samples were collected from each species (totally 20 samples). After DNA extraction, the fragment with 979 bp length from mitochondrial DNA was amplified using polymerase chain reaction. Sequencing was performed by automated Sanger methods then the obtained sequences were compared with sequences from other studies. The nucleotide sequences obtained were edited using the PHRED software (http://www.phrap.org /phredphrapconsed.html). After editing, basic local alignment search tool (BLAST) were used in order to find the homology of sequences. Further analysis of the sequences were carried out, by using the other software’s such as Chromas Lite (http://www.technelysium.com.au), Bio Edit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and the obtained sequences, were aligned with other COX3 gene of camel and other species, using CLC Main workbench 5.5 software (http://www.clcbio.com). The sequences were conducted using the maximum composite likelihood method by MEGA software (www.megasoftware.net, version v.5.2). Phylogenetic tree was constructed using the Neighbor-Joining method by the same software. Results and discussion The results shown that there are no differences between COX3 sequences in both samples from Iranian Camelus dromedaries and Camelus bactrianus and also their sequences have 100 percent homology with Camelus dromedarius (Arabian camel) and Iranian Camelus dromedaries Camelus bactrianus (Bactrian camel), respectively. Comparison of nucleotide and amino acid sequences and also three-dimensional structure of COX3 in Iranian camel species demonstrated that these two species have close genetic distance. Phylogenetic analysis revealed that these two species have the most genetic distance with Lama guanicoe among the CAMELIDAE FAMILY. The content of nucleotide sequences showed that the estimated frequencies of A + T and C + G were in the range of 52.4 and 47.6 percents for Iranian Camelus dromedaries and 53.2 and 46.8 percents for Iranian Camelus bactrianus, respectively. Conclusion COX3 sequence analysis shown that Iranian Dromedarius and Bactrianus camels had high level homology in sequence and nucleotide content.