Abstract: 【Objective】 An experiment was carried out in laboratory to study on expression of Avian reovirus σ3 gene in tobacco and its genicity reactions of the expression products. 【Method】 A pair of primers was designed according to the major antigen region of the σ3 gene derived from GenBank. A plant vector of pBI121-σ3 constitutively expressed σ3 gene was constructed. The pBI121-σ3 vector was transferred into the Agrobacterium strain EHA105 by thermal activation method and a recombinant Agrobacterium strain EHA105 containing pBI121-σ3 was obtained. After transformation of tobacco plants via Agrobacterium tumefaciens, the resistant plants were selected with kanamycin. The resistant plants were firstly analyzed by PCR, using σ3 gene specific primers, and then real-time fluorescence quantitative PCR was used to further estimate the copy number of the σ3 gene in positive plants, and endogenous RNR2 gene in tobacco was used as a reference gene. With a serial of dilutions, the standard curves of the cycle threshold (C_T) relative to the log of each initial template copy of σ3 and RNR2 genes were obtained. The transgenic copy number was obtained by comparing the initial template copy of σ3 gene with that of RNR2. Western blot analysis was used to examine the σ3 protein expression in transgenic tobacco. 【Result】 The recombinant plasmid was identified by restriction endonuclease analysis and by gene sequencing. It was confirmed by DNA sequencing that the recombinant plasmid pBI121-σ3 contains a complete σ3 gene, a correct insertion site and expected open reading frame (ORF). The plant nuclear vectors expressed the σ3 gene by using CaMV 35S promoter and NOS terminator, and a kanamycin resistance marker (NPT II). The σ3 gene may be expressed as a 61.6 ku green fluorescent fusion protein(GFP) in tobacco.  MS medium containing 350 mg cefotaxime·L^-1 inhibited the growth of Agrobacteria and 100 mg kanamycin·L^-1 can provide enough selective pressures. Eight resistant plants containing σ3 gene were obtained by PCR screening. The standard curve of RNR2 gene was expressed as y=-3.5352x+15.143, the standard curve of σ3 gene was expressed as y=-3.5366x+1.8265. Among the five putative transgenic lines, five had four copy numbers, whereas the negative control had none. Western blotting results showed that the 61.6 ku recombinant σ3 protein was successfully expressed, and the protein was specifically recognized by anti ARV positive serum. 【Conclusion】 A plant expression vector of pBI121-σ3 was successfully constructed and the regenerated transgenic σ3 gene tobacco plants were obtained. Recombinant σ3 protein has a good reactivity with anti ARV positive serum. The research findings provide a basis for further analysis on plants as bioreactors development and the production of σ3 oral vaccines.

Key words: avian reovirus, σ3 gene, plant expression vector, tobacco