| Porphyra yezoensis has become a basic and applied research model species in marine biology, owing to the high value of economic and scientific research. The fosmid library 0f Porphyra yezoensis was constructed.It contains 23,040 clones,and the recombination frequency is 100%. The most insert fragments are between 28 kb and 40 kb and the average insertion length is 35 kb. As a result, the library is 2.78 genome-equivalent coverage. Six clones were selected randomly to passage. After one hundred generation, the inset DNA fragment still exist. We designed primers with 18S, atpA, h2A, rbcL genes, and found one clone at least after several steps PCR. This study will lay the foundations for further understanding of molecular genetics features of Porphyra yezoensis.Mitogen-activated Protein Kinases is a serine / threonine protein kinase that widespread presence in eukaryotes. It is the end of cytoplasmic signal transduction pathway, and can phosphorylate a variety of protein including transcription factors, cytoskeletal proteins, protein kinase, and so in order to regulate the activity of them.It can do a great impact on gene expression, metabolism, cell division, cell morphology and cell survival. but the study of MAPK gene is still blank in in the big red alga.Two fosmid clones which contained two kind of MAPK gene were isolated from the fosmid DNA library of Porphyra yezoensis by PCR-pooling based on using partial known EST sequence. 2321bp和2189bp MAPK DNA sequence was cloned by sequence walking. We got full length PyMAPK1 gene containing an ORF of 1458 bp and one intron sequences(251 bp). The average GC content of the coding region of PyMAPK1 is 62. 96%, and The average GC content of the 245bp intron sequences is 55.38%, 7.58% lower than the coding region. The condon usage analysis found the third position of codon of PyMAPK1 had a strong codon usage bias towards C. The result of PyMAPK1 amino acid sequence analysis demonstrated PyMAPK1 protein possiblely contain 19α-helixes and 11β-strands and 11 Conservative domain of MAPK. The full length PyMAPK2 gene was cloned by sequence walking and the coding region of PyMAPK2 gene consists of 1515bp nucleotide acids. PyMAPK2 also has an intron sequences of 245bp. The average GC content of the coding region of PyMAPK2 is 64. 29%, and The average GC content of the 245bp intron sequences is 56.69%, 7.6% lower than the coding region. The condon usage analysis found the third position of codon of PyMAPK2 had a strong codon usage bias towards C. The result of PyMAPK2 amino acid sequence analysis demonstrated PyMAPK2 protein contain some functional sites, 19α-helixes , 10β-strands and 11 Conservative domain of MAPK. PyMAPK2 protein belonging to MAPK Group D which is plant specific, not only contain KD area (the regional characteristics of Group D), but also contain the CD area (Group D MAPK usually do not possess) . The analysis of PyMAPK2 protein phylogenetic trees showed that Group D MAPK has a separate evolution. PyMAPK1 containing TTY sequence, for a new type of MAPK protein. The analysis of PyMAPK1 protein phylogenetic trees showed that PyMAPK1 was different from other types of MAPK.The PyMAPK1 and PyMAPK2 expression of Porphyra yezoensis under the stress of mechanical trauma and ROS was monitored by real-time quantitative PCR. The results showed that the gene PyMAPK1 and PyMAPK2 in mechanical trauma stress has increased significantly. However, the increase in time and degrees were different. PyMAPK1 transcription increased slower and less than PyMAPK2. In ROS stress, PyMAPK1 transcription volume increased significantly, the increase early than in mechanical trauma stress. |
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