Two virus-encoded RNA silencing suppressors,P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

Functional analysis for gene silencing suppres- sor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- in- filtration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by ho- mologous sequence was strongly suppressed by the immix- ture infiltration of either the P14 or the S6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results sug- gested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.

Production of transgenic calves by somatic cell nuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.

Pedigree transmission disequilibrium test for QTL mapping of threshold trait

With biological and economic importance,threshold traits are discrete in phenotype but have the same polygenic genetic basis as quantitative traits. The traditional linkage analysis for quantitative traits is invalid for threshold traits due to their special characters. The transmission disequilibrium test (TDT) has received great attention recently in localizing human disease genes due to its simplicity and powerfulness. But TDT only deals with data from independent nuclear families and it will lose information about extended pedigree that incorporates information not only from parents and siblings but also from other relatives. The pedigree disequilibrium test (PDT) proposed by Martin in 2001 can be used to analyze the extended pedigree in human.In this study, PDT was introduced into the QTL mapping of threshold traits for farm animals, and was modified in order to accommodate the pedigree structures of farm animals.The modified PDT was renamed pedigree transmission disequilibrium test (PTDT) and its power and type I error were investigated and compared with that of PDT by Monte Carlo simulation. It was shown that PTDT is a robust and valid approach to mapping QTL of threshold trait. When the parental information is complete, PTDT and PDT are almost the same in terms of power and type I error. However, if the parental information is missing to a certain extent, PTDT is higher in power and lower in type I error than PDT. These results imply that PTDT can be a novel approach to QTL fine mapping of threshold traits based on the existing coarse mapping information.

Genome sequence variationanalysis of two SARS coronavirus isolates afterpassage in Vero cell culture

SARS coronavirus is an RNA virus whose rep- lication is error-prone, which provides possibility for escape of host defenses, and even leads to evolution of new viral strains during the passage or the transmission. Lots of varia- tions have been detected among different SARS-CoV strains. And a study on these variations is helpful for development of efficient vaccine. Moreover, the test of nucleic acid characterization and genetic stability of SARS-CoV is im- portant in the research of inactivated vaccine. The whole genome sequences of two SARS coronavirus strains after passage in Vero cell culture were determined and were com- pared with those of early passages, respectively. Results showed that both SARS coronavirus strains have high ge- netic stability, although nearly 10 generations were passed. Four nucleotide variations were observed between the sec- ond passage and the 11th passage of Sino1 strain for identi- fication of SARS inactivated vaccine. Moreover, only one nucleotide was different between the third passage and the 10th passage of Sino3 strain for SARS inactivated vaccine. Therefore, this study suggested it was possible to develop inactivated vaccine against SARS-CoV in the future.