Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
156
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: [email protected]
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
157
(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
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
158
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)
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
159
Marker 1 2 3 4 Marker
Primer pair
Hsp70-F
forward
Hsp70-R
reverse
ATGGCGAAAAACATGGCTATCGGC
CTAATCCACCTCCTCAATGGTGGGGCC
PCR product size = 1926 bp
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
160
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).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
161
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
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
162
************************************************************
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
************************************************************
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
163
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.
REFERENCES
1- Sharma, D. and Masison, D.C. (2009). Hsp70 structure, function, regulation and
influence on yeast prions. Protein Pept. Lett.16, 571-581.
2- Sharma, D. ; Martineau, C. N. ; Le Dall, M. T. ; Reidy, M. ; Masison, D. C. and
Kabani, M. (2009). Function of SSA subfamily of Hsp70 within and across
species varies widely in complementing Saccharomyces cerevisiae cell
growth and prion propagation. PloS one, 4(8): e6644.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
164
3- Kampinga, H.H. and Craig, E.A. (2010).The HSP70 chaperone machinery: J proteins
as drivers of functional specificity. Nat. Rev. Mol. Cell Biol.11(8): 579-592.
4- Pockley, A.G. and Multhoff,G. (2008). Cell stress proteins in extracellular fluids:
friend or foe? Novartis. Found. Symp.291: 86-95.
5- Pockley, A.G. ; Muthana, M. and Calderwood, S.K. (2008). The dual
immunoregulatory roles of stress proteins. Trends Biochem. Sci., 33:71-79.
6- Park, S. H. ; Bolender, N. ; Eisele, F. ; Kostova, Z. ; Takeuchi, J. ; Coffino, P. and
Wolf, D. H. (2007). The cytoplasmic Hsp70 chaperone machinery subjects
misfolded and endoplasmic reticulum import-incompetent proteins to
degradation via the ubiquitin–proteasome system. Molecular biology of the
cell, 18(1): 153-165.
7- Evdonin, A.L. ; Guzhova, I.V. ; Margulis, B.A. and Medvedeva, N.D. (2006).
Extracellular heat shock protein 70 mediates heat stress-induced epidermal
growth factor receptor transactivation in A431 carcinoma cells. FEBS Lett.
580(28): 6674-6678.
8- Njemini, R. ; Bautmans, I. ; Lambert, M. ; Demanet, C. and Mets, T. (2007). Heat
shock proteins and chemokine/cytokine secretion profile in ageing and
inflammation. Mechanisms of ageing and development, 128(7): 450-454.
9- Mayer, M.P. and Bukau, B. (2006). Hsp70 chaperones: Cellular functions and
molecular mechanism. Cell Mol Life Sci 62(6): 670-684.
10- Bhat, S. ; Kumar, P. ; Kashyap, N. ; Deshmukh, B. ; Dige, M.S. ; Bhushan, B. ;
Chauhan, A. ; Kumar, A. and Singh, G. (2016). Effect of heat shock protein
70 polymorphism on thermotolerance in Tharparkar cattle. Veterinary World,
9(2): 113-117.
11- Basirico, L. ; Morera, P. ; Primi, V. ; Lacetera, N. ; Nardone, A. and
Bernabucci, U.( 2011). Cellular thermotolerance is
associated with heat shock protein 70.1 genetic polymorphisms in Holstein
lactating cows. Cell Stress and Chaperones,16(4): 441–448.
12- Tamzil, M.H. ; Noor, R.R. ; Hardjosworo P.S. ; Manalu, W. and Sumantri, C.
(2013). Acute HS responses of three lines of chickens with different heat
shock protein (hsp)-70 genotypes. Int J Poult Sci 12(5): 264-272.
13- Gafer, J.A. ; Ghada H.A. and Rawash Z.M.(2015). Association of
Hsp70 Gene Polymorphism and Bull Semen Quality in
Winter and Summer Seasons. Alexandria Journal of
Veterinary Sciences,46 : 1-14. doi: 10.5455/ajvs.186038.
14- Singh, K. M. ; Singh, S. ; Ganguly, I. ; Nachiappan, R. K. ; Ganguly, A. ;
Venkataramanan, R. ... and Narula, H. K. (2017). Association of heat
stress protein 90 and 70 gene polymorphism with adaptability traits in Indian
sheep (Ovis aries). Cell stress & chaperones.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
165
15- Rosenkrans, J.C. ; Banks, A. ; Reiter, S. and Looper, M. ( 2010) .
Calving traits of crossbred brahman cows are associated with heat
shock protein 70genetic polymorphisms. Anim. Reprod., Sci.,
119(3): 178–182.
16- Cushman, R.A. (2013). Physiology and Endocrinology Symposium: the current status
of heat shock in early embryonic survival and reproductive efficiency. J Anim
Sci., 91(3):1141-1152. doi: 10.2527/jas.2013-6231.
17- Archana, P.R. ; Aleena, J. ; Pragna, P. ; Vidya, M.K. ; Niyas, A.P.A. ; Bagath, M.
; Krishnan, G. ; Manimaran, A. ; Beena, V. ; Kurien, E.K. ; Sejian, V.
and Bhatta, R.(2017). Role of Heat Shock Proteins in Livestock Adaptation
to Heat Stress. J Dairy Vet Anim Res., 5(1): 00127. doi:
10.15406/jdvar.2017.05.00127.
18- Habib, H.N. ; Hassan, A.F. and Khudaier, B.Y.(2017). Molecular Detection of
Polymorphism of Heat Shock Protein 70 (hsp70) in the semen of Iraqi
Holstein Bulls. Asian J. Anim. Sci., 11(3): 132-139.
19- Anju, M. ; Sonia, M. ; Balvinder, K. ; Khanna, S. and Sethi, R.K.
(2010). Evaluation of different methods of dna extraction from semen of
buffalo (bubalusbubalis) bulls. Buffalo Bulletin Vol.29 No.2.
20- Silva, E.C.B. ; Pelinca, M.A. ; Acosta, A.C. ; Silva, D.M.F. ; Gomes Filho, M.A.
and Guerra, M.M.P. (2014). Comparative study of DNA extraction
methodologies from goat sperm and its effects on polymerase chain reaction
analysis. Genetics and Molecular Research, 13 (3): 6070-6078.
21- Jinshan, Z. ; Lee, J. ; Kaidong, L. ; Nan, L. ; Ming, C. (2015). A pair of specific
recognition sheep hsp70.1 gene peptide and its coding gene and application.
Qingdao Institute of Animal Science and Veterinary Medicine.
patents/CN104745552A.
22- Clustal Omega. (2017). http://www.ebi.ac.uk/ /Tools/msa/clustalo/.
23- Pawar, N.H. ; Agrawal, R.K. ; Ramneek and Brah, G.S. (2013). Expression,
purification and characterization of recombinant Heat Shock Protein 70
(HSP70) from sheep and goat species. Int.J.Curr.Microbiol.App.Sci., 2(11):
440-452.
24- Banerjee, D. ; Upadhyay, R.C. ; Chaudhary, U.B. ; Kumar, R. ; ingh, S.V.;
Ashutosh, J. M. G. ; Polley, S. ; Mukherjee, A. ; Das, T.K. and De, S.
(2014). Seasonal variation in expression pattern of genes under HSP70
family in heatand coldadapted goats (Capra hircus). Cell Stress Chaperones,
19 (3): 401–408, doi.org/10.1007/s12192-013-0469-0.
25- Gade, N. ; Mahapatra, R. K. ; Sonawane, A. ; Singh, V. K. ; Doreswamy, R. and
Saini, M. (2010). Molecular Characterization of Heat Shock Protein 70-1
Gene of Goat (Capra hircus). Molecular biology international, vol. 2010.
104061/2010/ 108429.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
166
26- Hunt, R. C. ; Simhadri, V. L. ; Iandoli, M. ; Sauna, Z. E. and Kimchi-Sarfaty, C.
(2014). Exposing synonymous mutations. Trends in Genetics, 30(7): 308-321.
27- Nikbin, S. ; Panandam, J. M. ; Yaakub, H. ; Murugaiyah, M. and Sazili, A. Q.
(2014). Novel SNPs in heat shock protein 70 gene and their association with
sperm quality traits of Boer goats and Boer crosses. Animal reproduction
science, 146(3): 176-181.
28- Sangeeta, S., Arangasamy, A., Kulkarni, S., & Selvaraju, S. (2015). Role of amino
acids as additives on sperm motility, plasma membrane integrity and lipid
peroxidation levels at pre-freeze and post-thawed ram semen. Animal
reproduction science, 161, 82-88.
29- Barbato, V., Talevi, R., Braun, S., Merolla, A., Sudhakaran, S., Longobardi, S.,
& Gualtieri, R. (2017). Supplementation of sperm media with zinc, Daspartate
and co-enzyme Q10 protects bull sperm against exogenous oxidative
stress and improves their ability to support embryo development. Zygote,
25(2), 168-175.
30- Habib, H.N. ; Hassan, A.F. and Khudaier, B.Y.(2017). Association of hsp70 gene
Polymorphism with some of fresh semen characters in summer and winter to
Iraqi Holstein Bulls. I J S R. 78.46
