Bioinformatic Analysis On ESTs And Fd Gene Subunit Ⅰ Sequence From Sporophyte Of Porphyra Yezoensis Ueda

Porphyra yezoensis is one of the most economically important species of the world in the genus and is also an ideal material for genetics and molecular biology research. We partially sequenced one of the two libraries constructed with different methods used P. yezoensis sporophyte and bioinformatic analysis to the expressed sequence tags (EST) was performed. Finally, the Ferredoxin I subunit gene was cloned and analyzed based on the bioinformatic analysis result.The first cDNA library is constructed with the SMART method with a recombinant percentage of 88.2% and a titer of 1.52 × 10~~6pfu/ml before amplified, which ensures a good content to the genomic DNA and low frequency genes. And the second cDNA library is built with the Oligo-capping method with the purpose of pertaining the full-length of 5'end, which contains 1.8×10~6clones with the longest insert fragment of 3.5 K.There are 329 non-redundant EST groups clustered from the 719 expressed sequence tags (EST) sequenced from clones of the Oligo- capping cDNA library and among which the 60 strictest annotated unique genes are analyzed by the bioinformatic analysis method, i.e., Gene Ontology (GO), HMMPfam, and pathway analysis. The genes are classified from three aspects such as molecular function, biological process and cellular component with GO method and some domains such as COX1, SodFeC, GST_N, SHMT, and RNasePH which is essential to the function of enzymes and proteins in cells have been identified by HMMPfam, and the possible metabolism ways of the proteins or enzymes from the genes are detailed analyzed with pathway. The average GC content of the 329 unique genes is 53.0% and the third nucleotide of a codon has a higher GC content than that of the first and the second nucleotides, and P. yezoensis sporophyte is also found having strong codon usage bias which is similar to its leafy gametophyte. Similarity searches show a novelEST ratio of 60.2% and a new genes ratio of 81.8% in the present study which against the Porphyra ESTs Database, and thus it is necessary to conduct extensive studies to elucidate the characteristics of Porphyra functional genome.Ferredoxins are soluble iron-sulfur proteins found in various organisms with a prominent role to transfer electrons between enzymes, and a ferredoxin I subunit genomic gene is cloned from P. yezoensis sporophyte for the first time in the present study. The bioinformatic analysis shows that the genomic gene contains an GC rich intron from 280 to 306 after the ATG site within the 918 nucleotides of the gene and its cDNA includes a coding region of 588 bp for 195 amino acids with a stem loop structure from nt 861 to 865 including the possible poly asenylation signal at the 3'-UTR, and the four cysteine residuals which are essential to the protein function are at the 81, 94, 113 and 132 positions, respectively. The GC content of the gene coding region is 63.3% which is 5 percent higher than that of the whole sequence and 10 percent higher than the averaged GC content of the P. yezoensis sporophyte genomic DNA. The amino acid sequence of the Fd gene is aligned with three kinds ferredoxin and some conservative residues such as CPHL and DL are found.