Sui Zhenghong

Bioinformatic analysis of expressed sequence tags from sporophyte of Porphyra yezoensis (Bagiaceae, Rhodophyta)∗

Abstract A total of 719 expressed sequence tags (EST) clustered into 329 non - redundant EST groups are obtained from the sporophte cDNA library of red alage, Porphyra yezoensis. Gene Ontology (GO) analysis is employed in characterizing 60 strictest annotatd unique genes out of the 329 EST groups and some domains such as COXI, Sod_Fe_C, GST_N, SHMT, and RNase_PH related to the enzymes and proteins functioning in cells have been identified by HMMPFAM search. As its leafy gametophyte, the similar codon usage with storong bias is found in P. yezoensis filamentous sporophyte, regardless of some differences found in given amino acids. The average GC content of the 329 unique genes is 53.0 %. In contrast, the third nucleotide of codon exhibits a higher GC content (72 %) than that of the first (58 % ) and the second (42 %) nucleotides. Similarity search of the present study shows a noves EST ratio of 60.2 % Which is against the Porphyra ESTs database, suggesting further investigations towards elucidating the cha...

Identification of phase and sex-related ISSR markers of red algaGracilaria lemaneiformis

Six ISSR primers are employed to display the polymorphism of different phases and sexes of red algaGracilaria lemaneiformis, and two of them, P1 and P3, amplified distinct band patterns. The ISSR pattern amplified by primer P1 of the female gametophyte is identical to that of tetrasporophyte, but distinct from that of male gametophyte. Of the bands produced by primer P3, one is specific to female gametophyte. Three morphologically similar fronds can be easily identified using ISSR technique. Two specific markers, SM1 and SF3, related to male gametophyte and female gametophyte, are cloned and sequenced. The homologous sequences of SM1 are found to encode a hypothetical protein. There is no homologous sequence of SF3 that can be found in GenBank.

Application of RAPD in genetic diversity study onGracilaria lemaneiformis III. Phase and sex related markers

Random amplification of polymorphic DNA (RAPD) was conducted by using 10 random primers (P-1 to P-10) inGracilaria lemaneiformis. Phase and sex specific bands were amplified by primers P-2, P-6, P-7 and P-8: for P-2 a 1.4 kb band was found in female gametophytes and tetrasporophytes, for P-6, a 0.6 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.76 kb band appeared in male gametophytes and tetrasporophytes; for P-7, a 0.72 kb band appeared in female gametophytes and tetrasporophytes; for P-8, a 0.73 kb band only appeared in male gametophytes.

Expression of the Phycoerythrin Gene of Gracilaria lemaneiformis (Rhodophyta) in E.coli and Evaluation of the Bioactivity of Recombinant PE

Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) (P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mg mL−1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.

Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. A cytosolic GAPDH encoding gene (gpd) ofGracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme ofG. lemaneiformis had high homology with those of seven red algae. The 5′-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site ofCyanidium caldarium was different from those of the other six algae at the last two residues (CL to LF), thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH ofG. lemaneiformis might have undergone an evolution similar to those ofPorphyra yezoensis, Chondrus crispus, andGracilaria verrucosa. C. caldarium had a closer evolutionary relationship withCyanidioschyzon merolae than withCyanidium sp. Virtual Northern blot analysis revealed thatgpd ofG. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis ofG. lemaneiformis.

Cloning and analysis of phycoerythrin genes inGracilaria Lemaneiformis (Rhodophyceae)

Cloning and sequencing of the genes coding for the α- and β- subunit of phycoerythrin (PE) of a red alga—Gracilaria lemaneiformis (GL) are reported. Alignment of 1084 nucleotides sequenced with three known red algal PE genes,Rhodella violacea (RV),Polysiphonia boldii (PB) andAglaothamnion neglectum (AN), showed high level of conservation, and similarities of 77,6% (between GL and RV), 77.9% (GL and AN) and 79.0% (GL and PB). The similarities of amino acids were 84.8% (between GL and RV), 85.7% (GL and PB), and 80.6% (AN and GL), higher than those among nucleotides.