Molecular characterization of heat shock protein 70 gene in Iraqi buffalo

The heat shock protein 70 (HSP 70) has important roles in protecting cells and keeping them alive when exposed to different stress conditions. The polymorphism of the hsp 70 gene could be linked with the ability of stress tolerance. This study aimed to determine the polymorphism of the hsp 70 gene in Iraqi buffaloes and study bits effects on the resistance to stress. This study was conducted during from November 2018 to February 2019. The number of buffalo females used was 35 at the age of 4 6 years, which belonged to the local farmers from Basra city, Iraq. The DNA was extraction from the blood samples then the polymerase chain reaction (PCR) amplification was performed. The DNA sequences were analyzed by using bioinformatics analysis. The results of the molecular analysis showed that there were two groups of the hsp 70 gene as a compare with the same genes in GenBank due to silent and missense mutations. Based on these findings, it can be concluded that the Iraqi buffaloes have adapted to the surrounding environmental conditions as a result of the action of HSP 70 proliferation. The hsp 70 gene was a useful biomarker of stress tolerance in buffaloes.


Introduction
The HSP 70 is a dominant protein member of the huge HSP family. This family has a diversity of functions in the cells such as protecting living cells under the different conditions of stress (1). Furthermore, the HSP 70 was specified to be a confirmatory molecular marker for determination the response of different stress conditions in the farm animals (2). The hsp 70 gene can be used as an elect gene for the election cattle based on the stress leniency traits (3). The hsp 70 gene is imperative to generate HSP 70 that influencing on the stress conditions (4). The HSP 70 has a molecular weight between 68 -73 KDa (5), composed 641 amino acid (6) and the coding region of the hsp 70 gene in buffalo with length 1926bp (7). Despite the HSP 70 is consider as a highly conserved polypeptide (8), the polymorphisms of the hsp 70 gene explain the variance between individuals in resistance to the different stress conditions. On one hand, the previous studies have reported the diversity of polymorphisms of the hsp 70 gene (9)(10)(11)(12)(13)(14)(15), as well as linked some of the different production traits (16)(17)(18)(19)(20). On the other hand, the variations in the DNA sequences are of value because they might change the interaction of the HSP 70, thus, enhancing the animal's response to the stress conditions (21). Buffaloes have significant economic importance of the different food industry in many developing countries of Asia that regards as one of the most important sources of milk and meat production (22). Iraq has the second largest number of buffaloes after Egypt in the Middle East region, it is also ranked globally as the 12 th of buffalos milk (23). In spite of the vital importance to buffaloes in Iraq, the environmental stress such as high temperatures (24), and the water salinity (25) have negatively affected on the production of buffalo. Therefore, there is an urgent need to look for mechanisms to resist the stress conditions and to select animals with a higher capacity to resist these conditions. The most important molecular mechanisms that the body possesses are the family of HSPs, especially HSP 70. It has important roles in protecting cells and keeping them alive when exposed to the different stress conditions (26), and the possibility of using its polymorphisms as an electoral marker to resist stress conditions (27).
Little is known about the adaptation mechanism of local Iraqi buffaloes for surviving in stress conditions and there is a lack of studies on this subject. Therefore, the current study was designed to determine the polymorphisms of the hsp 70 gene in the Iraqi buffaloes and study its role on the living cells in environmental stress.

Animal and Sampling
This study was conducted during from November 2018 to February 2019. The number of the samples collected from buffalo females aged between 4-6 years old were 35. All the samples were selected randomly at different stages of the milk production which belonged to local farms in Basra city, Iraq. The blood samples were collected from the jugular vein using sterile tubes with size 10 ml containing 0.5 ml EDTA solution as an anticoagulant, all samples were kept frozen at -20 ºC till the DNA extraction process.

DNA extraction
The DNA was extracted from the blood samples using Genomic DNA Kit (Geneaid Biotech, Taiwan). Then it was estimated the concentration and the purity of DNA by using NanoDrop as described by Desjardins and Conklin (28).

PCR Amplification
The amplification reaction was 25 μl containing 1.0 μl DNA template (75 ng), 1.0 μl Forward primer (10 µM), 1.0 μl Revers primer (10 µM), 12.5 μl of 2 X PCR master mixes and 9.5μl water nuclease free. According to APICAL lab (formerly named First BASE Laboratories) Malaysia, the primer was used to amplify conserved coding region of the HSP 70 promoter (Table 1). PCR condition and thermal cycling protocols were summarized in Table 2. To detect PCR product was used 1.5% ethidium bromide 0.5 μg/ml stained agarose gel.

Results
The ratio of 260/280 was between 1.80 to 1.85 for all samples. PCR product size was 1926 bp (Figure 1). Based on the analysis multiple sequence alignment, two different polymorphisms were obtained according to the mutations as compared to reference sequence (Accession number EU099315.1). They were divided in to Group 1 and Group 2.

Group 1
The position 235 (G>C) mutation, the position 364 (A>T) a missense mutation that occurred as a result of the change of amino acid from methionine to leucine. The position1079 (A>G), the position 1080 (C>T) both are missense mutations that occurred as a result of the change of amino acid from asparagine to serine. The position 1354 (G>A) a missense mutation that occurred as a result of the change of amino acid from aspartic acid to asparagine. The position 1638 (C>G) silent mutation and the position 1639 (T>A) a missense mutation that occurred as a result of the change of amino acid from phenylalanine to isoleucine (Record in GenBank by accession number LC496272).

Group 2
The position 35 (G >C) a missense mutation that occurred as a result of the change of amino acid from glycine to alanine. The position 156 (C>T) a missense mutation that occurred as a result of the change of amino acid from glycine to arginine. The position 235 (G>C) silent mutation, the position 364 (A>T) a missense mutation that occurred as a result of the change of amino acid from methionine to leucine, position 562 (A>T) silent mutation, the position 563 (C>T)a missense mutation that occurred as a result of the change of amino acid from threonine to leucine. The position1079 (A>G) and the position 1080 (C>T) are both missense mutations that occurred as a result of the change of amino acid from asparagine to serine. The position 1354 (G>A) is a missense mutation that occurred as a result of the change of amino acid from aspartic acid to asparagine. The position 1638 (C>G) is silent mutation and the position 1639 (T>A) is a missense mutation that are occurred as a result of the change of amino acid from phenylalanine to isoleucine. All the mutations were clarified in figure 2, and the changes of amino acids were clarified in figure 3. The phylogenetic tree analysis ( Figure  4) showed very close affinity with all sequences of the Bubalus bubalis hsp 70 gene. However, it did not match another sequence, but they were closer to complete the hsp 70 gene in Bubalus bubalis of water buffalo (Accession Number EU099315.1)

Discussion
The ratio of 260/280 was the best ratio for the purity of the DNA as mentioned (28). The results are in line with what (7,19), noting the possibility of polymorphism for the hsp 70 gene in Buffalo, but the results did not match 100% with any pre-recorded GeneBank sequence, including the complete gene of the hsp 70 gene to Bubalus bubalis (Accession number EU099315.1). The corresponding ratio in the first group and the second group were 99.64 and 99.43% (30) respectively. This finding suggests that the Iraqi Buffaloes have different polymorphisms of the hsp 70 gene. The silent mutations can alter the capacity of an mRNA to code for protein via impact the average of the translation, by the change in codon usage through the production of budding protein (31). As for the missense mutations can be molecular markers to resist the heat stress (32,33). This Genetic diversity may give Iraqi buffalo the ability to withstand various stress conditions (34). Moreover, the polymorphism in the coding region of the hsp 70 gene is associated with an advantage of longevity and survival, as well as the polymorphisms are correlated with increased heat tolerance (35). Nevertheless, the result of the Phylogenetic tree analysis is supporting the hypothesis that the Iraqi buffalo have adapted to environmental conditions, because these genetic changes can determine the ability of animals to withstand different stress conditions (7).

Conclusion
In conclusion, the current study showed two different groups of the hsp 70 gene, these differences may refer to the diversity of animals' ability to tolerate different stress conditions. Therefore, more studies to investigate the role of the polymorphism of the hsp 70 gene and the different production traits in the Iraqi buffalo are needed.