Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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MOLECULAR DETECTION OF POLYMORPHISM OF HEAT
SHOCK P ROTEIN 70 (hsp70) IN THE SEMEN OF ARABI RAMS
Hassan Nima Habib*, Bassam Yasein Khudaier** ,Amad Falah Hassan*
* College of Agriculture, University of Basrah, Basra, Iraq
** College of Veterinary, Medicine University of Basrah, Basrah, Iraq
Key words: Molecular detection, Arabi rams, heat stress.
Corresponding Author: fllan8@yahoo.com
ABSTRACT
Heat shock proteins Hsp70 proteins belong to the heat shock protein family, boost cells
survival in hostile environments, involved in many cellular biological processes. The
polymorphism of hsp70 gene relates to the possibility of tolerating different stress
conditions. This study was carried out during 1st April to 31th June 2016, 20 Arabi Rams,
known fertility, 2 – 2.5 years old, from the Agricultural Research Station / College of
Agriculture / University of Basra, Iraq, were used, Semen was collected by using the
artificial vagina, after training well. DNA extracted then Polymerase chain reaction(PCR)
amplified sequencing, BLAST analysis and multiple sequence alignment were carried out.
The results showed two haplotypes, haplotype G1 , haplotype G٢ compared with same
gene in GenBank, all mutations were silent except the mutation is site 514 was missense
appeared in G1. It concluded that the Arabi rams in Iraq have new polymorphism of
hsp70 gene which can help cope with difficult environmental conditions.
INTRODUCTION
Heat shock protein (HSPs) are a highly preserved polypeptides, lodging almost in
any cell, they were classified according to their molecular weights, one of their most
important roles is as like molecular chaperones and are decisively Participate in
maintaining protein homeostasis and cell survival (1). Hsp70 is a member of HSPs
family, which are spread in all living organisms, whether eukaryotic or prokaryotic (2,3),
which in addition to working as molecular chaperones, have immune functions( 4,5), but
its main role is folding the proteins and prevent the improper assembly of proteins (6).
Hsp70 has a significant role in protecting cells against cellular stress including heat stress
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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(7), by promoting cells tolerance to environmental changes and pathologic conditions (8).
The work of the Hsp70 can be summarized in three main axes in all organisms, prevent of
ingathering of proteins, corrected folded proteins that are exposed to misfolded and
dissolve and refolding of aggregated proteins under the stress conditions (9). These
functions of HSP70 as a molecular chaperone and cell protect versus heat stress, making it
able to denaturing proteins (10). On the other hand many studies have indicated a
relationship between the polymorphism of hsp70 gene and heat tolerance in Holstein cow
(11), chicken (12), in buffalo (13) and in sheep (14).The polymorphism of hsp70 gene
also affects reproductive performance of Farm Animal males, by reducing the various
stress conditions on the animals (15,16). Therefore, the hsp70 gene is an ideal biological
marker to resist heat stress in farm animals (17). No previous study in Iraq has been
conducted on the polymorphism of hsp70 gene in the semen of Arabi Rams in Iraq,
except for one study on the polymorphism of the hsp70 gene in Iraqi Holstein Bulls by
(18), therefore no information about the polymorphism of hsp70 gene in Arabi Rams.
This study aimed to identify the polymorphism of hsp70 gene in Arabi Rams.
MATERIALS AND METHODS
Animals and semen collection
Twenty Arabi Rams, known fertility, 2 – 2.5 years old, from the Agricultural
Research Station / College of Agriculture / University of Basra, Iraq, were used in the
present study. During 1st April to 31th June 2016. Semen collected from Rams by using
the artificial vagina method, specialized artificial vagina for sheep (after training well).
DNA extraction
DNA was extracted from semen samples using Chelex ( Sigma Aldrich, USA) as
described by (19). Concentration and purity of DNA was estimated by using Nanodrop
system (Nano Drop thermo scientific 200, USA). 1.8 was adopted as the best purity of
260/280 ratio (20)
PCR amplification and Sequencing
Amplifications were performed according to First BASE Laboratories / Malaysia,
in 25 μl reactions (Table 1), to amplify conserved region within the hsp70 promotor
,primer was used as in table 2 , the PCR amplification cycling protocols of PCR
amplification as in table 3. PCR product was detected on 1.5 % ethidium bromide stained
agarose gel, wih size 1926pb approximately (Fig 1), as described (18).
PCR products Purified and sequenced in First BASE Laboratories / Malaysia .
(www.base-asia.com) . Nucleotides sequence alignments, were done using Geneious
version 10.1.3 software. BLAST Analysis was carried out on website
(www.ncbi.nlm.nih.gov. ) Multiple Sequence Alignment and compared with same gene in
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Gene Bank, accession number NM_001267874.1, were carried out on website
( http://www.ebi.ac.uk/Tools/msa/clustalo/. ).
(1) Table
PCR
Reaction Components
Table (2) primer
Table (3) Thermal Cycling Protocol of PCR
First BASE Laboratories (2016)
Components Amount (μl)
Water, nuclease free 9.5
2X PCR Master Mix 12.5
Forward primer, 10 μM 1
Reverse primer, 10 μM 1
DNA template (75 ng) 1
TOTAL 25
First BASE Laboratories (2016)
Cycle step Temp (° C) Time Number of Cycles
Initial Denaturation 95 5 min 1
30
Denaturation 94 30 s
Annealing 61 30 s
Extension 72 2 min
Final Extension 72 10 min ١
First BASE Laboratories (2016)
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Marker 1 2 3 4 Marker
Primer pair
Hsp70-F
forward
Hsp70-R
reverse
ATGGCGAAAAACATGGCTATCGGC
CTAATCCACCTCCTCAATGGTGGGGCC
PCR product size = 1926 bp
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RESULTS AND DISCUSSION
The results of nucleotides sequence analysis and the Multiple Sequence Alignment
( MSA) compared with heat shock protein hsp70 gene of sheep in the gene bank, suggest
a presence two new haplotypes (Figure2) , classified as two groups :
First group : haplotype A : showed no match at 100% with other nucleotides sequence
of other group or in Gene Bank heat shock protein hsp70 gene, the nucleotide in position
1308, T was substituted by the nucleotide C (T <C), a silent mutation that did not
encoding a new amino acid, 10 rams were found in this group.
Second group: haplotype B : in comparison with the first group and the same gene in
Gene Bank, the nucleotide C was substituted with the nucleotide G (C <G), a missense
mutation that resulted in a change in the amino acid encoding (Histidine change to
Aspartic) in the position 514, a mutation not previously recorded in sheep except in
Chinese sheep by (21) , registered as a patent with ID CN104745552A but did not match
the results of the current study. In addition, nucleotide G was substituted by nucleotide A
(G <A) in the position 1524, a silent mutation, 10 rams were found in this group.
The (MSA) of hsp70 gene showed that the ratio of A and B haplotypes were 99.89% and
99.84% respectively with the same gene in the Gene Bank, accession number
NM_001267874.1 (22 ), these results are consistent with Pawar et al., (23) and Banerjee
et al., (24) in the possibility of polymorphisms of hsp70 gene in sheep, but the results are
not consistent with any previous study in the position of mutations, and may be due to the
fact that the current study is the first of its kind and was not preceded by any other study.
These mutations in hsp70 gene may refers to the development of this protein through the
positive election among domestic animals (25).
On the other hand the silent mutations may effect on the role of translated protein through
diverse cellular mechanisms (26), and it lead to polymorphism to the hsp70 gene, that’s
associated with the semen quality (27).
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As for the missense mutation that occurred in haplotype B due to the change of Histidine
amino acid to the Aspartic, it may improve the quality of semen (28, 29 , 30).
NM_001267874.1 ATGGCGAAAAACATGGCTATCGGCATCGACCTGGGCACCACCTACTCCTGCGTGGGGGTG 60
G1 ------------------------------------------TACTCCTGCGTGGGGGTG 18
G2 ------------------------------------------TACTCCTGCGTGGGGGTG 18
******************
NM_001267874.1 TTCCAGCACGGCAAGGTGGAGATCATCGCCAACGACCAGGGCAACCGCACCACCCCCAGC 120
G1 TTCCAGCACGGCAAGGTGGAGATCATCGCCAACGACCAGGGCAACCGCACCACCCCCAGC 78
G2 TTCCAGCACGGCAAGGTGGAGATCATCGCCAACGACCAGGGCAACCGCACCACCCCCAGC 78
************************************************************
NM_001267874.1 TACGTGGCCTTCACCGATACCGAGCGGCTCATCGGCGATGCAGCCAAGAACCAGGTGGCG 180
G1 TACGTGGCCTTCACCGATACCGAGCGGCTCATCGGCGATGCAGCCAAGAACCAGGTGGCG 138
G2 TACGTGGCCTTCACCGATACCGAGCGGCTCATCGGCGATGCAGCCAAGAACCAGGTGGCG 138
************************************************************
NM_001267874.1 CTGAACCCGCAGAACACCGTGTTCGACGCGAAGCGGCTGATCGGCCGCAAGTTCGGCGAC 240
G1 CTGAACCCGCAGAACACCGTGTTCGACGCGAAGCGGCTGATCGGCCGCAAGTTCGGCGAC 198
G2 CTGAACCCGCAGAACACCGTGTTCGACGCGAAGCGGCTGATCGGCCGCAAGTTCGGCGAC 198
************************************************************
NM_001267874.1 CCGGTGGTGCAGTCGGACATGAAGCACTGGCCTTTCCGCGTGATCAACGACGGAGACAAG 300
G1 CCGGTGGTGCAGTCGGACATGAAGCACTGGCCTTTCCGCGTGATCAACGACGGAGACAAG 258
G2 CCGGTGGTGCAGTCGGACATGAAGCACTGGCCTTTCCGCGTGATCAACGACGGAGACAAG 258
************************************************************
NM_001267874.1 CCTAAAGTGCAGGTGAGCTACAAGGGGGAGACCAAGGCGTTCTACCCAGAGGAGATCTCG 360
G1 CCTAAAGTGCAGGTGAGCTACAAGGGGGAGACCAAGGCGTTCTACCCAGAGGAGATCTCG 318
G2 CCTAAAGTGCAGGTGAGCTACAAGGGGGAGACCAAGGCGTTCTACCCAGAGGAGATCTCG 318
************************************************************
NM_001267874.1 TCGATGGTGCTGACCAAGATGAAAGAGATCGCCGAGGCGTACCTGGGCCACCCGGTGACC 420
G1 TCGATGGTGCTGACCAAGATGAAAGAGATCGCCGAGGCGTACCTGGGCCACCCGGTGACC 378
G2 TCGATGGTGCTGACCAAGATGAAAGAGATCGCCGAGGCGTACCTGGGCCACCCGGTGACC 378
************************************************************
NM_001267874.1 AACGCGGTGATCACCGTGCCGGCCTACTTCAACGACTCGCAGCGGCAGGCCACCAAGGAC 480
G1 AACGCGGTGATCACCGTGCCGGCCTACTTCAACGACTCGCAGCGGCAGGCCACCAAGGAC 438
G2 AACGCGGTGATCACCGTGCCGGCCTACTTCAACGACTCGCAGCGGCAGGCCACCAAGGAC 438
************************************************************
NM_001267874.1 GCGGGGGTGATCGCGGGGCTGAACGTGCTGAGGATCATCAACGAGCCCACGGCCGCCGCC 540
G1 GCGGGGGTGATCGCGGGGCTGAACGTGCTGAGGATCATCAACGAGCCCACGGCCGCCGCC 498
G2 GCGGGGGTGATCGCGGGGCTGAACGTGCTGAGGATCATCAACGAGCCCACGGCCGCCGCC 498
************************************************************
NM_001267874.1 ATCGCCTACGGCCTGCACCGGACGGGCAAGGGGGAGCGCAACGTGCTCATCTTTGACCTG 600
G1 ATCGCCTACGGCCTGCACCGGACGGGCAAGGGGGAGCGCAACGTGCTCATCTTTGACCTG 558
G2 ATCGCCTACGGCCTGGACCGGACGGGCAAGGGGGAGCGCAACGTGCTCATCTTTGACCTG 558
*************** ********************************************
NM_001267874.1 GGCGGGGGCACGTTCGACGTGTCCATCCTGACGATCGACGACGGCATCTTCGAGGTGAAG 660
G1 GGCGGGGGCACGTTCGACGTGTCCATCCTGACGATCGACGACGGCATCTTCGAGGTGAAG 618
G2 GGCGGGGGCACGTTCGACGTGTCCATCCTGACGATCGACGACGGCATCTTCGAGGTGAAG 618
************************************************************
NM_001267874.1 GCCACGGCCGGGGACACGCACCTGGGCGGGGAGGACTTCGACAACAGGCTGGTGAACCAC 720
G1 GCCACGGCCGGGGACACGCACCTGGGCGGGGAGGACTTCGACAACAGGCTGGTGAACCAC 678
G2 GCCACGGCCGGGGACACGCACCTGGGCGGGGAGGACTTCGACAACAGGCTGGTGAACCAC 678
************************************************************
NM_001267874.1 TTCGTGGAGGAGTTCAAGAGGAAGCACAAGAAGGACATCAGCCAGAACAAGCGGGCCGTG 780
G1 TTCGTGGAGGAGTTCAAGAGGAAGCACAAGAAGGACATCAGCCAGAACAAGCGGGCCGTG 738
G2 TTCGTGGAGGAGTTCAAGAGGAAGCACAAGAAGGACATCAGCCAGAACAAGCGGGCCGTG 738
************************************************************
NM_001267874.1 AGGCGGCTGCGCACGGCGTGCGAGCGGGCCAAGAGGACCTTGTCGTCCAGCACCCAGGCC 840
G1 AGGCGGCTGCGCACGGCGTGCGAGCGGGCCAAGAGGACCTTGTCGTCCAGCACCCAGGCC 798
G2 AGGCGGCTGCGCACGGCGTGCGAGCGGGCCAAGAGGACCTTGTCGTCCAGCACCCAGGCC 798
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************************************************************
NM_001267874.1 AGCCTGGAGATCGACTCCCTGTTCGAGGGCATCGACTTCTACACGTCCATCACCAGGGCG 900
G1 AGCCTGGAGATCGACTCCCTGTTCGAGGGCATCGACTTCTACACGTCCATCACCAGGGCA 858
G2 AGCCTGGAGATCGACTCCCTGTTCGAGGGCATCGACTTCTACACGTCCATCACCAGGGCA 858
***********************************************************
NM_001267874.1 CGGTTCGAGGAGCTGTGCTCCGACCTGTTCCGGAGCACCCTGGAGCCGGTGGAGAAGGCT 960
G1 CGGTTCGAGGAGCTGTGCTCCGACCTGTTCCGGAGCACCCTGGAGCCGGTGGAGAAGGCT 918
G2 CGGTTCGAGGAGCTGTGCTCCGACCTGTTCCGGAGCACCCTGGAGCCGGTGGAGAAGGCT 918
************************************************************
NM_001267874.1 CTACGCGACGCCAAGCTGGACAAGGCCCAGATCCACGACCTGGTCCTGGTGGGGGGCTCC 1020
G1 CTACGCGACGCCAAGCTGGACAAGGCCCAGATCCACGACCTGGTCCTGGTGGGGGGCTCC 978
G2 CTACGCGACGCCAAGCTGGACAAGGCCCAGATCCACGACCTGGTCCTGGTGGGGGGCTCC 978
************************************************************
NM_001267874.1 ACCCGCATCCCCAAGGTGCAGAAGCTGCTGCAGGACTTCTTCAACGGGCGCGACCTCAAC 1080
G1 ACCCGCATCCCCAAGGTGCAGAAGCTGCTGCAGGACTTCTTCAACGGGCGCGACCTCAAC 1038
G2 ACCCGCATCCCCAAGGTGCAGAAGCTGCTGCAGGACTTCTTCAACGGGCGCGACCTCAAC 1038
************************************************************
NM_001267874.1 AAGAGCATCAACCCGGACGAGGCGGTGGCATACGGGGCGGCGGTGCAGGCGGCCATCCTG 1140
G1 AAGAGCATCAACCCGGACGAGGCGGTGGCATACGGGGCGGCGGTGCAGGCGGCCATCCTG 1098
G2 AAGAGCATCAACCCGGACGAGGCGGTGGCATACGGGGCGGCGGTGCAGGCGGCCATCCTG 1098
************************************************************
NM_001267874.1 ATGGGGGACAAGTCGGAGAACGTGCAGGACCTGCTGCTGCTGGACGTGGCTCCCCTGTCG 1200
G1 ATGGGGGACAAGTCGGAGAACGTGCAGGACCTGCTGCTGCTGGACGTGGCTCCCCTGTCG 1158
G2 ATGGGGGACAAGTCGGAGAACGTGCAGGACCTGCTGCTGCTGGACGTGGCTCCCCTGTCG 1158
************************************************************
NM_001267874.1 CTGGGACTGGAGACGGCCGGAGGCGTGATGACCGCCCTGATCAAGCGCAACTCCACCATC 1260
G1 CTGGGACTGGAGACGGCCGGAGGCGTGATGACCGCCCTGATCAAGCGCAACTCCACCATC 1218
G2 CTGGGACTGGAGACGGCCGGAGGCGTGATGACCGCCCTGATCAAGCGCAACTCCACCATC 1218
************************************************************
NM_001267874.1 CCCACGAAGCAGACGCAGATCTTCACCACCTACTCGGACAACCAGCCGGGCGTGCTGATC 1320
G1 CCCACGAAGCAGACGCAGATCTTCACCACCTACTCGGACAACCAGCCGGGCGTGCTGATC 1278
G2 CCCACGAAGCAGACGCAGATCTTCACCACCTACTCGGACAACCAGCCGGGCGTGCTGATC 1278
************************************************************
NM_001267874.1 CAGGTGTACGAGGGCGAGAGGGCCATGACTCGGGACAACAACCTGCTGGGGCGCTTCGAG 1380
G1 CAGGTGTACGAGGGCGAGAGGGCCATGACCCGGGACAACAACCTGCTGGGGCGCTTCGAG 1338
G2 CAGGTGTACGAGGGCGAGAGGGCCATGACTCGGGACAACAACCTGCTGGGGCGCTTCGAG 1338
***************************** ******************************
NM_001267874.1 CTGAGCGGCATCCCGCCGGCCCCGCGGGGGGTGCCCCAGATCGAGGTGACCTTCGACATC 1440
G1 CTGAGCGGCATCCCGCCGGCCCCGCGGGGGGTGCCCCAGATCGAGGTGACCTTCGACATC 1398
G2 CTGAGCGGCATCCCGCCGGCCCCGCGGGGGGTGCCCCAGATCGAGGTGACCTTCGACATC 1398
************************************************************
NM_001267874.1 GACGCCAATGGCATCCTGAACGTCACGGCCACGGACAAGAGCACGGGCAAGGCCAACAAG 1500
G1 GACGCCAATGGCATCCTGAACGTCACGGCCACGGACAAGAGCACGGGCAAGGCCAACAAG 1458
G2 GACGCCAATGGCATCCTGAACGTCACGGCCACGGACAAGAGCACGGGCAAGGCCAACAAG 1458
************************************************************
NM_001267874.1 ATCACCATCACCAACGACAAGGGCCGGCTGAGCAAGGAGGAGATCGAGCGCATGGTGCAG 1560
G1 ATCACCATCACCAACGACAAGGGCCGGCTGAGCAAGGAGGAGATCGAGCGCATGGTGCAG 1518
G2 ATCACCATCACCAACGACAAGGGCCGGCTGAGCAAGGAGGAGATCGAGCGCATGGTGCAG 1518
************************************************************
NM_001267874.1 GAGGCGGAGAAGTACAAGGCAGAGGACGAGGTCCAGCGCGAGAGGGTGTCTGCCAAGAAC 1620
G1 GAGGCGGAGAAGTACAAGGCAGAGGACGAGGTCCAGCGCGAGAGGGTGTCTGCCAAGAAC 1578
G2 GAGGCAGAGAAGTACAAGGCAGAGGACGAGGTCCAGCGCGAGAGGGTGTCTGCCAAGAAC 1578
***** ******************************************************
NM_001267874.1 GCGCTGGAGTCGTACGCCTTCAACATGAAGAGCGCCGTGGAGGATGAGGGGCTGAAGGGC 1680
G1 GCGCTGGAGTCGTACGCCTTCAACATGAAGAGCGCCGTGGAGGATGAGGGGCTGAAGGGC 1638
G2 GCGCTGGAGTCGTACGCCTTCAACATGAAGAGCGCCGTGGAGGATGAGGGGCTGAAGGGC 1638
************************************************************
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NM_001267874.1 AAGATCAGCGAGGCGGACAAGAAGAAGGTGCTGGACAAGTGCCAGGAGGTGATTTCCTGG 1740
G1 AAGATCAGCGAGGCGGACAAGAAGAAGGTGCTGGACAAGTGCCAGGAGGTGATTTCCTGG 1698
G2 AAGATCAGCGAGGCGGACAAGAAGAAGGTGCTGGACAAGTGCCAGGAGGTGATTTCCTGG 1698
************************************************************
NM_001267874.1 CTGGACGCCAACACCTTGGCGGAGAAGGACGAGTTTGAGCACAAGAGGAAGGAGCTGGAG 1800
G1 CTGGACGCCAACACCTTGGCGGAGAAGGACGAGTTTGAGCACAAGAGGAAGGAGCTGGAG 1758
G2 CTGGACGCCAACACCTTGGCGGAGAAGGACGAGTTTGAGCACAAGAGGAAGGAGCTGGAG 1758
************************************************************
NM_001267874.1 CAGGTGTGTAACCCCATCATCAGCAGACTGTACCAGGGGGCGGGCGGCCCCGGGGCTGGC 1860
G1 CAGGTGTGTAACCCCATCATCAGCAGACTGTACCAGGGGGCGGGCGGCCCCGGGGCTGGC 1818
G2 CAGGTGTGTAACCCCATCATCAGCAGACTGTACCAGGGGGCGGGCGGCCCCGGGGCTGGC 1818
************************************************************
NM_001267874.1 GGCTTTGGGGCTCAGGCCCCTAAAGGGGGCTCTGGGTCTGGCCCCACCATTGAGGAGGTG 1920
G1 GGCTT------------------------------------------------------- 1823
G2 GGCTT------------------------------------------------------- 1823
*****
NM_001267874.1 GATTAG 1926
G1 ------ 1823
G2 ------ 1823
G1: Haplotype A
G2: Haplotype B
NM_001267874.1 : gene of hsp70 in GenBank
Fig. 2: Identified hsp70 gene in Arabi rams and the same gene in GenBank CLUSTAL
Omega (1.2.4) multiple sequence alignment
CONCLUSION
This study was showed two haplotypes of hsp70 gene in the Arabi rams in Iraq, due
to the occurrence of mutations in more than one location, suggesting that genotypes of
hsp70 could have been genetically modified to adapt to environmental conditions.
ACKNOWLEDGMENTS
The authors would like to thank Prof. Dr: Asaad Y. Ayied Department of Animal
Production, College of Agriculture, University of Basrah, Iraq. For his scientific
assistance in completing this paper.
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