[Sequence analysis of the coat protein gene of Chinese soybean mosaic virus strain SC7 and comparison with those of SMV strains from the USA].

To unveil genetic variations between the predominant soybean mosaic virus (SMV) strains in China and in the USA, as well as to reveal the potential relevance between the similarity of gene sequences and the virulence of the viruses, we isolated and sequenced the coat protein (CP) gene of Chinese SMV strain SC7 by RT-PCR and compared the SC7 sequence with those of SMV strains from the USA. Analysis is showed that the CP gene of SC7 was 795 nucleotides in length and encoded 265 in amino acids'. The CP gene of SC7 and those of the strains from the USA exhibited 4%-5% nucleotide diversity and 1%-2% diversity amino acids. The conserved amino-acid sequence associated with aphid spread in the USA strains was DAG, and corresponded to DAD in SC7. The virulence of SC7 was greater than that of the SMV strains from the USA. Nevertheless, no clear relationships between sequence similarity of the CP genes from different strains and their virulence on differential hosts were found.

Sequence level analysis of recently duplicated regions in soybean [Glycine max (L.) Merr.] genome.

A single recessive gene, rxp, on linkage group (LG) D2 controls bacterial leaf-pustule resistance in soybean. We identified two homoeologous contigs (GmA and GmA') composed of five bacterial artificial chromosomes (BACs) during the selection of BAC clones around Rxp region. With the recombinant inbred line population from the cross of Pureunkong and Jinpumkong 2, single-nucleotide polymorphism and simple sequence repeat marker genotyping were able to locate GmA' on LG A1. On the basis of information in the Soybean Breeders Toolbox and our results, parts of LG A1 and LG D2 share duplicated regions. Alignment and annotation revealed that many homoeologous regions contained kinases and proteins related to signal transduction pathway. Interestingly, inserted sequences from GmA and GmA' had homology with transposase and integrase. Estimation of evolutionary events revealed that speciation of soybean from Medicago and the recent divergence of two soybean homoeologous regions occurred at 60 and 12 million years ago, respectively. Distribution of synonymous substitution patterns, K(s), yielded a first secondary peak (mode K(s) = 0.10-0.15) followed by two smaller bulges were displayed between soybean homologous regions. Thus, diploidized paleopolyploidy of soybean genome was again supported by our study.

[Short hairpin RNA targeting vascular endothelial growth factor effectively inhibits expression of vascular endothelial growth factor in human retinal pigment epithelium].

OBJECTIVE: To design shRNA targeted to human vascular endothelial growth factor (VEGF) and to evaluate the effect of VEGF. shRNA on expression of VEGF in human retinal pigment epithelium (RPE) cells in vitro. METHODS: Human RPE cells were isolated with enzyme-assisted microdissection. The cells were identified by immunohistochemical method with antibody to cytokeratin and S-100. Plasma DNA was identified via restriction enzyme EcoRI and SamI. shRNAs (P1, P2) specific for human VEGF were designed. DNA expression vector is pSilencer 4.1-CMV of Ambion company. P3 is negative control nonspecific shRNA. There are 5 groups. Group 1: VEGF in cultured human RPE exposed to 100 micromol/L CoCl2 30 h; Group 2: VEGF in cultured human RPE in normal culture medium; Group 3, 4, 5: VEGF in cultured human RPE exposed to 100 micromol/L CoCl2 30 h after P1, P2, P3 transfection, respectively. VEGF level in conditioned media was measured by Western blot. RESULTS: The cells in culture could be stained with both cytokeratin and S-100 antibodies. The length of two fragment was 3.3 kb and 1.6 kb, respectively, which indicated that the extraction and purification were successful. The expression of VEGF in RPE was increased significantly (P < 0. 001) in group 1 as compared with group 2. Hypoxia-induced upregulation of human VEGF is halted by siRNA application in vitro (P < 0. 001 and P < 0. 001 in group 3 and 4 compared with group 1, respectively). shRNAs targeted hVEGF effectively and specifically inhibited hypoxia-induced VEGF levels in human RPE. The level of VEGF was reduced 65.9% and 52.4% in groups 3 and 4, respectively. There was no difference between group 5 and 1 (P = 0. 147). There was no difference of beta-actin production in RPE cells among groups. CONCLUSIONS: Delivery of shRNA can be used in vitro to target specific RNAs of VEGF and to reduce the level of the specific protein product (VEGF) in the targeted cells (human RPE). This work established the basis for the using of RNA interference in studies of retinal biology and for the treatment of a variety of retinal angiogenic diseases, especially the choroidal neovascularization.