农杆菌介导轮状病毒抗原蛋白VP4基因遗传转化花生的研究

Transgenetic Research of Antigen VP4 Gene into Peanut (Arachis hypo-gaea L.) via Agrobacterium tumefaciens

选用鲁花14与花育23花生品种,通过农杆菌介导开展了轮状病毒抗原蛋白VP4基因遗传转化研究,从转化植株中随机选取26株表现Kan抗性植株进行nptII基因的PCR检测,结果有22株能扩增出620bp左右的nptII基因条带,阳性率约为84.62%。提取nptII基因显示为阳性的植株叶片DNA作模板,用VP4基因特异引物进行PCR扩增,经琼脂糖凝胶电泳分析,所有nptII基因阳性的植株均扩增出了约2350bp的特异性条带,而野生型没有。对部分转基因植株进一步进行PCR-Southern杂交和Southern杂交分析,发现转基因植株中出现了阳性杂交信号,表明VP4基因的确已经整合到花生的基因组中,并且是1-2个拷贝。用RT-PCR分析了11株转G1VP4基因的植株,证明插入鲁花14中的VP4基因已经正常转录,利用Westernblot方法检测筛选到的4株转基因花生,分别提取其蛋白,在30kD处出现特异性蛋白条带,这为获得转基因创新种质材料奠定了基础。

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 α-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 expressionof the rotavirus VP4 antigen in transgenic peanuts is a critical factor in the development of a safe and effective rotavirus vaccine.