Molecular Cloning And Bioinformatics Analysis Of Prohibitin From Porphyra Yezoensis

Prohibitin is a conserved protein, which was first found in mammals. The results of somestudies indicated that prohibitin exists widely in organisms and it is a much conserved protein.Therefore, the protein has drawn researcher’s great attention because of its potential functionsuch as anti-cell proliferation, interfere cell senescence, apoptosis and other biologicalprocesses. In this dissertation, the results of cloning and bioinformatics analysis of aprohibitin gene from P. yezoensis was reported.First of all, some ESTs of prohibitin from Japan P. yezoensis database were searched,selected and DNA fragment assembled. We got an1176bp DNA sequence. A pair of specificprimers were designed according to the assembled DNA sequence. An1126bp DNA fragmentwas cloned by PCR with cDNA of P. yezoensis cell total RNA as template and specificprimers. BLAST results showed that the DNA fragment has high sequence similarity ofprohibitins from other species. The DNA fragment contains a complete open reading frame ofprohibitin, an ORF with894bp.Secondly, the physical and chemical properties and structural features of deducedprohibitin protein of P. yezoensis were analyzed and predicted by using bioinformaticssoftware and tools. Predictive analysis showed that cloned prohibitin (PyPHB) is an unstablehydrophilic protein containing297amino acids. The molecular weight is32954.5Da. Itsisoelectric point is6.88. In the secondary structure, the most abundant elements is α-helix. Itsproportion is56.23%. The accounting random coil is24.24%, the extended chain is16.16%,and the β-angle is3.37%. The possibility of subcellular localization of PyPHB in cytoplasm ismaximum69.6%, in nuclear is17.4%, in mitochondria is8.7%, and in cytoskeleton is4.3%.In addition, there is neither transmembrane region no signal peptide in PyPHB amino acidsequence. But PyPHB contains phosphorylation sites of Ser, Thr and Tyr, and multiplefunctional sites, where the most possible function is transcription.Finally, a phylogenetic tree was constructed to analyze the genetic relationship ofPyPHB and prohibitins from different species. In NCBI, through BLAST we collected40assumed PHB amino acid sequences, covering mammals, angiosperms, amphibians and fungiand constructed a phylogenetic tree using MEGA4.0method. The results showed thatPorphyra yezoensis is closely related to Chondrus crispus, another red seaweed.