Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5thInternational Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
20
INVESTIGATION THE POLYMORPHISM OF BONE
MORPHOGENETIC PROTEIN 15 (BMP-15) GENE IN IRAQ
COW.
Hayder Abdul-Kareem Hasan AL-Mutar
Department of Surgery and Obstetrics , College of Veterinary Medicine,– University
of Baghdad ,Baghdad, Iraq
Keywords: BMP-15 gene, cattle, polymorphism.
ABSTRACT
Bone Morphogenetic Protein 15 (BMP-15) was known to regulate functions of ovary
in mammals. The aim of this research was tocompare the BMP15 expression of
ovaries incow and calf todetermine a relationship between the level of BMP15 genes
and the developmental low competence of calfoocytes.Oocyte collected from 15cattle
and 10 calves. Extracted DNA by intron kit (Korea).The BMP15 expression in
Oocytes surrounded completely andpartiallyby cumulus cells of adult ovaries was
higher significantly than that incalf ovaries. Oocytepolymorphism in protein calves
compared with adults
INTRODUCTION
BMP15 geneis X-linked in oocytes expressed involved in granulose cell
regulation and differentiation by mitosis granulose cell, repression follicle stimulating
hormone receptor expression and engaged in the stimulation of kit ligand expression,
All of which contributing a significant role in female fertility in mammals (1,2).
Currently the hypothesis based on the non-covalently bond homo and hetero dimers of
the proteins BMP15 regulated the fertility in bovine (3). The biological roles of Bone
Morphogenetic Protein 15 (BMP15) are not completely understood even if gene to
regulate function of granulose cells, and the BMP15play an important role in the
process of follicular development and oocyte maturation (4). BMP15 known as
prolificacy candidate genes, play key roles in regulating ovarian functions in animals,
BMP15 mRNAs in cumulus granulose cells can be used as molecular markers
forpredicting oocyte developmental potential.(1,5).
MATERIALS AND METHODS
Cattles ovaries were collected from Al-shulla slaughter, from 10 calves, 9-11
months and from 15 cattle,2-5 years. Oocyte recovery from follicles by aspiration
methods (6,7). DNA was extracted by using the standard protocol by intron kit
procedure. After extraction of genomic DNA, gel electrophoresis was used to detect
the presence and integrity of the extracted DNA and presence of PCR product. Two
conserved primers (forward primer: 5'-CTCTGAGACCAAACCGGGTA -3' and
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5thInternational Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
21
reverse primer: 5'-CATGCCACCAGA ACTCAAGA-3') (3). thermal cycling
conditions were done as follows: Denaturation at 95 °C for 5 min, followed by 35
cycles of 94 °C for 35s, 55°C for 35s and 72 °C for 35s with final incubation at 72 °C
for 10 min using a thermal Cycler (Gene Amp, PCR system 9700; Applied
Biosystem). The Polymerase chain reaction (PCR) products were extracted by 2%
agarose gel electrophoresis and visualized by contact with ultra violet light
(302nm).Sequence of nucleotides of BMP-15 gene by using BioEdit program, which
was performed by National Instrumentation Center for Environmental Management
(NICEM) online at (http://nicem. Snu .ac. kr/main/ ?en _ skin =index.html),
biotechnology lab, machine is DNA sequencer 3730XL, Applied Biosystem),
Homology look for was conducted using Basic Local Alignment Search
Device(BLAST) program which was available at the National Center Biotechnology
Information (NCBI) online at (http://www.ncbi.nlm.nih.gov ).Data were submitted to
statistical analysis using chi-Square test, spss program (8).
RESULT AND DISCUSSION
The analysis of BMP-15 gene polymorphism was carried out using PCR
method. Genomic DNA of cattle was successfully amplified by pair of primer that
covers entire coding sequence of BMP-15 gene. Genomic DNA of white blood cells
was also used for amplification of BMP-15 gene using PCR specific primers. The
amplified fragment which is yielded of single band of the desired product with a
molecular weight of 350 base pair appeared sharp in agarose gel through Gel
electrophoreses technique and loaded with (100-1000bp) DNA ladder (figure 1).
Figure (1): The product was electrophoresis on 2% agarose gel at 5 volt/cm2, 1x TBE buffer for 2
hours. M: DNA ladder (100-10000bp), Lane 1-7 product for GnRHR gene of adult cow and Lane
8-15 product of calf, PCR product of band size 350bp. visualized under U.V light after staining
with red stain safe.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5thInternational Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
22
The sequencing of amplified product of BMP-15 gene from calf , out of them appeared
98% compatibility with standard Bostaurus breed bone morphogenetic protein 15
(BMP15) gene from 5054 to 5301 number of nucleotide from gene of gene Bank
results as shown in Figure (2), Sequence ID: gb|EU712722.1|, and have number score
(236) bits.
bone morphogenetic protein 15, partial [Bostaurus]
Sequence ID: emb|CAD58882.1|Length: 96Number of Matches: 1
Range 1: 7 to 88GenPeptGraphicsNext MatchPrevious Match
Score Expect Method Identities Positives Gaps Frame
165 bits(418) 8e-52 Compositional matrix adjust. 80/82(98%) 80/82(97%) 0/82(0%) +3
Query 3 RHQLHLTHSHLSCHVEPWVQKSPTNHFPSSGRGSSKPSLLPKTWTEMDIMEHVGQKLWNH
182
RHQLHLTHSHLSCHVEPWVQKSPTNHFPSSGRGSSKPSLLPKWTEMDIMEHVGQKLWNH
Sbjct 7 RHQLHLTHSHLSCHVEPWVQKSPTNHFPSSGRGSSKPSLLPKAWTEMDIMEHVGQKLWNH 66
Query 183 KGRRVLRLRFVCQQPRGSEVLE 248
KGRRVLRLRFVCQQPRGSEVE
Sbjct 67 KGRRVLRLRFVCQQPRGSEVRE 88
R H Q L H L T H S H L S C H V E P W V Q K S P T N H F P S S G R
G S S K P S L L P K T W T E Met D I Met E H V G Q K L W N H K G
R R V L R L R F V C Q Q P R G S E V L E S G
Bostaurus bone morphogenetic protein 15 (BMP15) gene, exons I, II and partial cds
Sequence ID: gb|EU712722.1|Length: 5829Number of Matches: 1
Range 1: 5054 to 5301GenBankGraphicsNext MatchPrevious Match
Score Expect Identities Gaps Strand
431 bits(233) 4e-117 243/248(98%) 0/248(0%) Plus/Plus
Query 1 ACCGCCATCAGCTTCACCTAACTCATTCCCACCTCTCCTGCCATGTGGAGCCCTGGGTCC
60
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5054 ACCGCCATCAACTTCACCTAACTCATTCCCACCTCTCCTGCCATGTGGAGCCCTGGGTCC
5113
Query 61 AGAAAAGCCCAACCAATCACTTTCCTTCTTCAGGAAGAGGCTCCTCAAAGCCTTCCCTGT
120
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5thInternational Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5114 AGAAAAGCCCAACCAATCACTTTCCTTCTTCAGGAAGAGGCTCCTCAAAGCCTTCCCTGT
5173
Query 121 TGCCCAAAACTTGGACAGAGATGGATATCATGGAACATGTTGGGCAAAAGCTCTGGAATC
180
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5174 TGCCCAAAGCTTGGACAGAGATGGATATCATGGAACATGTTGGGCAAAAGCTCTGGAATC
5233
Query 181 ACAAGGGGCGCAGGGTTCTACGACTCCGCTTCGTGTGTCAGCAGCCAAGAGGTAGTGAGG
240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 5234 ACAAGGGGCGCCGGGTACTACGACTCCGCTTCGTGTGTCAGCAGCCAAGAGGTAGTGAGG
5293
Query 241 TTCTTGAG 248
|||||||
Sbjct 5294 TTCGTGAG 5301
Figure (2): Sequencing of sense flanking the BMP-15 gene, for cases of calf, obtained
from Gene Bank. Query represents of sample; Sbject represent of database of National
Center Biotechnology Information (NCBI).
Table 1: Type of polymorphism and amino acid change in sense of BMP-15 gene in
cattle's.
location of
gene bank
Nucleotide
change
Amino acid change Predicted
effect
Type of
mutation
A5064 G CAA>CAG Glutamine> Glutamine Silent Transition
G5182A GCT>ACT Alanine>Threonine Missense Transition
C5245A CGG>AGG Arginine> Arginine Silent Transversion
A5250T GTA>GTT Valine>Valine Silent Transversion
G5297T CGT>CTT Arginine>Leucine Missense Transition
In total cases of cattle's had two type of transversion substitution in location
5245 C>A, 5250 A>T and three types of transition substitution in 5064 A>G, 5182
G>A and 5297 G>T, of BMP15 gene shown as in table 1. In this research, data
showed the presence ofbovine BMP15 DNA and protein expression,and occur
polymorphism of calf BMP15 compare adult in cumulus oocyte may be showed
differences inpatterns of BMP15 incumulus cells and oocytes between cow and calf
ovariesrelated to the follicle atresia status or to the estrous cycle (9).There was no
difference in the oocyteBMP15 between calf and cow, and was smallerin calf
cumulus cells than in cow cumulus cells(10).In follicles isgreater in cows than in
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5thInternational Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
24
calves(11,12). The lower developmentalcompetence of calf oocytes may be partially
explainedby a deficiency of BMP15 in cumulus cells(13).(14)Evaluating transcript
abundance of bovine oocytes of different developmental competence found no
difference in BMP15 gene expression. The oocyte can store histone mRNA and
proteins during oogenesis (15,16). Additionally, studies have reported that a
relationship exists between the amount of histones and oocytes competence in bovine.
BMP15 were positively associated with age (17). BMP15 can stimulateoocyte
development (18)The BMP15 genes contain two exons separated by a single intron
that encode a rough endoplasmic reticulum signal peptide. The signal peptide region
was encoded by the first exon, the proregion by segments of both exons and the
mature peptide region by the second exon (19).Oocytewas a major source of both
BMP15 and GDF9 and the major target for these factors was the granulose cells (20).
فی الابقار العراقیة BMP- التحری عن التغایرات الوراثیة لجین 15
حیدر عبد الکریم حسن المطر
کلیة الطب البیطری، فرع الجراحة والتولید، جامعة بغداد،بغداد ،العراق.
الخلاصة
یعرف بتنظیموظائفالمبیضفیالثدییات . الھدف من ھذا البحث ھو مقارنة التغایرات الوراثیة فی (BMP15)
بین العجول الصغیرة والابقار وتحدید العلاقة بین مستوى ھذه الجینات ونخفاض BMP المبایض للجین 15
تطور البیوض فی العجلات الصغیرة . تم جمع البیوض من 15 بقرة و 10 عجلات . وتم استخلاص الحامض
النووی بستخدام کت الانترون (کوری المنشاء) . التغایرات الوراثیة للجین فی البویضات المحاطة بشکل کلی
وجزئی فی خلایا الرکمة المبیضیة فی الابقار تکون بنسبة اعلى من العجلات الصغیرة . التغایرات الوراثیة
البروتینیة فی العجلات اعلى منھا فی الابقار الکبیرة.
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