ⅠIdentification And Complete Nucleotide Sequence Analysis Of Wisteria Vein Mosaic Virus ⅡCloning, Expression And Activity Analysis Of A Trypsin Inhibitor Gene From Populus Alba Var.Pyramidalis Bge

The mosaic symptom of wisteria (Wisteria sinensis) in Beijing is caused by a potyvirus. The Beijing isolate of the potyvirus was mechanically transmitted to C. amaranticolor, C. quinoa, N. clevelandii and V. faba, producing local chlorotic lesions, systemic chlorotic lesions, mildly systemic chlorotic lesions and systemic chlorotic lesions, respectively. Although the virus coat protein was present in the endosperms of the seeds of infected wisteria plants by ELASA tests, it is not seed-borne. The virus isolate was serologically related to Bean common mosaic virus (BCMV) and Watermelon mosaic virus (WMV), the molecular weight of its CP subunit was confirmed to be 35.0 kD by SDS-PAGE and Western blot analysis. Cells of V. faba leaves infected with the virus had cytoplasmic pinwheel inclusion bodies typical of potyviruses. The above biological properties and sequence analysis of the coat protein gene and 3' non-translated region (NTR) showed that this potyvirus is Wisteria vein mosaic virus (WVMV). This is the first report of WVMV occurrence in China.The genomic sequence of WVMV-BJ was determined by sequencing 7 overlapping viral cDNA clones generated by RT-PCR, which represents the first complete sequence for the virus. The sequence is 9695 nucleotides in length excluding the 3' terminal poly (A) tail and contains a single open reading frame of 9279 nucleotides encoding a large polyprotein of 3092 amino acids with predicted Mr of 353.4 kD. The genome of WVMV-BJ has a 164 nt 5'-non coding and a 252 nt 3'-non coding region. The size of the genome and the encoded polyprotein is in agreement with other potyviruses and contains nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. The P1 and P3 were the most variable proteins while CI, NIb and CP were relatively conserved.The CP gene of WVMV-BJ was amplified by RT-PCR, and ligated to the expression vector pET22b (+). The recombinant plasmid pET-WVMVCP was transformed into E. coli BL21 (DE3) and then induced by IPTG to express a fusion protein. It was shown by SDS-PAGE and Western blot analysis that the CP gene was expressed at high level. The molecular weight of the fusion protein was about 34.4kD. An antiserum with high specificity was produced after the rabbit was immunized with purified fusion protein, and its titer was determined to be 1/1024 by micro-precipitation, or 1/8192 by ACP-ELISA.To explore novel ways for controlling potyviruses by inhibiting the proteinases encoded by this group of viruses, a proteinase inhibitor gene was cloned and its protein product expressed for test of its inhibitory activity to some proteinases. A Kunitz trypsin inhibitor gene (PT1) was isolated from mechanically wounded poplar (Populus alba var. pyramidalis Bge) leaves by RT-PCR. The longest open reading frame in the PTI gene, which contains conventional 'TATA' and 'CCAAT' transcription control elements, potentially encode a peptide of 213 amino acids. Sequence comparisons showed that PTI was most closely related to PtTI2 and PtT11 from trembling aspen, with identities of 95% and 98%,respectively. The PTI gene was expressed in E. coli to produce a fusion protein and the purified product was shown to have inhibitory activity to bovine trypsin in vitro. Western blot analysis showed that the fusion protein of 27.5 kD strongly react with the antibody raised against PtTI2.