关键词: 花生, 子叶, 遗传转化, VP4基因

Abstract: Peanut (Arachis hypogaea L.) is an important commercial crop worldwide and provides an excellent source of protein and other nutrients. So it is one of the ideal vectors in oral vaccine of transgenic plant. The Agrobacterium strain LBA4404 harboring the binary vector pBI121 was used for transformation. The plasmid carries genes for VP4 (2 350 bp) of the human rotavirus (HRV) driven by CaMV 35S promoter and neomycin phosphotransferase (npt II). Two peanut cultivars were used as source of cotyledon explants in this experiment. Efficient transformation of cotyledons by A. tumefaciens strain LBA4404 carrying nptII and VP4 gene on binary vectors led to the production of a large percentage of transgenic plants. Transformed individuals were obtained through selection on medium containing 125 mg L-1 Kan. Integration of the transgenes was assessed by PCR amplification and Southern blot hybridizations. Taking pBG1VP4 or pBG1VP4 plasmid as positive control, non-transformed peanut as negative control, 22 plants among 26 plants grown up through selection on medium containing 125 mg L-1 Kan were assessed by PCR amplification of 620 bp fragment of npt II gene. Then all of 22 plants were assessed by PCR amplification of 2 350 bp fragment of VP4 gene. Taking VP4 gene with a-32P-dCTP maker as probe, five plants selected randomly from 22 positive plants were analysed by PCR-Southern blot hybridizations and showed DNA bloting bands. Then the genomic DNA of 4 plants chosen from PCR-Southern positive plants was further analyzed with Southern blot hybridizations and showed correspondent DNA blotting bands. The results showed that the foreign gene was integrated into genome of transformated peanuts. The total RNA from 11 plants of Luhua 14 was assessed by RT-PCR analysis and evidenced the expression of G1VP4 gene. In addition, expression of critical protein in 30 kD was assayed with Western-blot method. An edible vaccine based on the VP4 of human rotavirus (HRV) could provide a means to protect children from severe acute diarrhea, enabling intact antigen to reach the gut associated lymphoid tissue, as the rigid walls of the plant cell protect antigenic proteins from the acidic environment of stomach. Elevated expression of the rotavirus VP4 antigen in transgenic peanuts is a critical factor in the development of a safe and effective rotavirus vaccine.

Key words: Peanut, Cotyledon, Genetic transformation, VP4 gene