signature=0ee587a54b50fee67711bf84ba60fa37,Molecular Study on Environmental Stress (Heat Shock- Gene...

摘要：

Degenerate primers are particularly useful in amplifying homologous genes from different species. The present study aimed to investigate the importance of degenerate primers and the HSP70 family signature to create a new specific motif for HSP70 proteins family and Described a method for designing degenerate primers for a given multiple alignment of DNA sequences of HSP70 gene family using Clustalw algorithm. An Insilco approach was used to find a homology between more than one Accession numbers of DNA sequences, (X67711.2) was for Oryza sativa (HSP70), (AY372071.1) was for Nicotiana tabacum (HSP70) and (L41253.2) was for Lycopersicon esculentum (Hsc70), the three accession numbers were retrieved by the BLASTn program depend on their expected value (E-value). Multiple sequence alignment was performed by clustalw algorithm to produce a conserved blocks and determined the consensus region was used to produce the forward and reverse primer by the primer select module of DNA STAR LASER GENE 7.0. An Insilico PCR module of FASTPCR program ver.4.0.8 was performed to detect the melting temperatures (Tm) and Predicted the PCR product size. The results of deigned degenerate primer showed that there was a homology found between the designed primers and the DNA templates for the previous three accession numbers with at least 80% identity. The result of degenerate PCR showed that the three bands of the amplified PCR products of the three accession numbers were detected at the same molecular weight of (385bp) with a difference about 15 bp compared to the Insilco PCR product (385 bp). Degenerate PCR was used to isolate the consensus coding sequence of HSP70 gene family for Nigella sativa and sequenced the predicted band. The results of degenerate PCR showed that the amplified PCR products for the three accession numbers and Niegella sativa PCR products were detected at the same molecular weight ( bp) and the result of Nigella sativa, sequenced band (385bp) was justified and corrected to be 345bp showed a homology to HSP70 gene family and recorded with a new accession number (HM803244) in the NCBI. The bioinformatics data were retrieved from the curated databases of protein to detect the conserved sequences among the records of HSP70. A new motif was built and all the statistical analysis of the new motif was processed to build the dendogram based on the physiochemical properties of amino acid sequences. A new specific motif was designed by PRATT tool which depended on the multiple sequence alignment algorithm and by using position specific iterative BLAST (PSI-BLAST). The new specific motif was used to construct the polygenetic tree from multiple sequence alignment for every taxonomy of (bacteria, viridiplantae and metazoa) and the single peptide motif was converted to predict the 3-D structure. The 3-D structure templates in the PDB (Protein Data Bank) using (scanprosite) database search. The result of the predicted 3-D motif showed a highly similarity and stabilization to the other crystallography templates of HSP70 in different organisms.

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