Li HuaPing

Sequence analysis of segment 8 of five Chinese isolates of Rice gall dwarf virus and expression of a main outer capsid protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8 (S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%–98.8% and 97.3%–99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%–95.6% and 95.0%–96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta (DE3) II cells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.

Qualitative detection assay for transgenic papaya with replicase gene of papaya ringspot virus.

A set of transgenic detection methods and technology had been established to supervise and manage the commercial producing and selling of the transgenic papaya-Huanong No.1 Papain(Pn)was chosen as the endogenous comparison genes of papaya and primers were designed to detect the transgenic papaya and non-transgenic papaya.The regular PCR was established to detect the PRSV-Rep,exogenous CaMV 35S promoter sequence,NOS terminator sequence and the NPTⅡ of transgenic papaya.The clear bands were amplified when the annealing temperature was 40 ℃ to 60 ℃.The sensitivity of the single gene was 5×10-7 g.The Triple-gene-PCR detection system was created to detect three combination genes of NPTⅡ,Rep and CaMV 35S;or NPTⅡ,Rep and Nos;or NPTⅡ,Rep and Papain.And the Quad-PCR system was built to test four genes of NPTⅡ,Rep,CaMV35S and Nos as well.