First insights into the large genome of Epimedium sagittatum (Sieb. et Zucc) Maxim, a Chinese Ttaditional medicinal plant.

Epimedium sagittatum (Sieb. et Zucc) Maxim is a member of the Berberidaceae family of basal eudicot plants, widely distributed and used as a traditional medicinal plant in China for therapeutic effects on many diseases with a long history. Recent data shows that E. sagittatum has a relatively large genome, with a haploid genome size of ~4496 Mbp, divided into a small number of only 12 diploid chromosomes (2n = 2x = 12). However, little is known about Epimedium genome structure and composition. Here we present the analysis of 691 kb of high-quality genomic sequence derived from 672 randomly selected plasmid clones of E. sagittatum genomic DNA, representing ~0.0154% of the genome. The sampled sequences comprised at least 78.41% repetitive DNA elements and 2.51% confirmed annotated gene sequences, with a total GC% content of 39%. Retrotransposons represented the major class of transposable element (TE) repeats identified (65.37% of all TE repeats), particularly LTR (Long Terminal Repeat) retrotransposons (52.27% of all TE repeats). Chromosome analysis and Fluorescence in situ Hybridization of Gypsy-Ty3 retrotransposons were performed to survey the E. sagittatum genome at the cytological level. Our data provide the first insights into the composition and structure of the E. sagittatum genome, and will facilitate the functional genomic analysis of this valuable medicinal plant.

Characterization and vaccination of two novel Schistosoma japonicum genes screened from a cercaria cDNA library.

Two novel genes, SJCWL05 and SJCWL06, were harvested from screening of Schistosoma japonicum (S. japonicum) cercaria cDNA library by using pig sera vaccinated (VPS) with S. japonicum immature egg ws-vaccine (S. japonicum iEw). Prokaryotic recombinant plasmids pGEX-4T-1/SJCWL05 and pGEX-4T-1/SJCWL06 were constructed to analyze their immunogenicity, which was confirmed by SDS-PAGE and Western blotting. Two eukaryotic recombinant plasmids, pcDNA3/SJCWL05 and pcDNA3/SJCWL06, were constructed, and their ability to protect mice against challenge of S. japonicum was evaluated. All mice vaccinated with pcDNA3/SJCWL05 or pcDNA3/SJCWL06 developed ELISA-specific anti-S. japonicum SIEA (S. japonicum soluble immature egg antigens) antibody. Immunoprotection experiments showed that worms and liver eggs reduced 34.64% and 39.14% in the pcDNA3/SJCWL05 group and those reduced 27.17% and 27.95% in the pcDNA3/SJCWL06 group, respectively. The reduction rates of intestine and uterine eggs in female worms of both groups reached 39.45% and 38.5% as well as 30.02% and 28.7%, respectively. Results of our study suggest that novel genes, SJCWL05 and SJCWL06, are potential vaccine candidates against schistosomiasis japonica.

[Advances of gene enrichment in plant genome].

The genome size varies greatly in higher plants. Repetitive sequences account for most of the large plant genomes while low-copy or single copy genic sequences, referred to as gene space, take up only a small portion of the genomes. Considering the large amount of repetitive sequences, it is a great challenge to obtain genic sequences using high-throughout methods in non-model plants bearing large genomes. Currently, several approaches have been developed for gene enrichment on a genome-wide scale, such as cDNA library, methylation filtration library, high Cot library and transposon tagging. Here, we reviewed the technical principles, advantages and disadvantages of these methods, as well as the recent development of methylation filtration technology. An in-depth discussion was performed for selection of one method or combination of methods according to the research objectives and plant materials, especially for plants with large genomes.

[Cloning and expression of the succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum Chinese strain in E. coli].

OBJECTIVE: To clone the full-length gene encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) Chinese strain and express it in Escherichia coli. METHODS: According to the published incomplete EST (BU804141) of SjSDISP and the sequence of multiclone sites of lambda gt11 vector, 2 pairs of primers were designed and synthesized. Then the 3' and 5'ends of the EST of the SjSDISP from adult Schistosoma japonicum cDNA library were amplified by anchored PCR. After sequencing, a full-length cDNA sequence of the SjSDISP was obtained, and then it was cloned into prokaryotic expression vector pGEX-4T-1. Identified by agarosed gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for the expression under the temperature-dependent condition and Western blot analysis. RESULTS: A 1,071 bp sequence was obtained. Sequence analysis showed that the fragment contained a complete open reading frame (ORF), encoding 278 amino acid residues. This target fragment was cloned into the prokaryotic expression vector pGEX-4T-1, and expressed in Escherichia coli. SDS-PAGE revealed that the molecular weight of the expressed fusion recombinant product was 56 kD. Western blot showed that the recombinant protein was recognized by polyclonal rabbit antiserum immunized with Schistosoma japonicum adult worm antigen. CONCLUSION: Cloning of the full-length gene encoding SjSDISP and its bacterial expression were successfully done.