Production of Transgenic Soybean to Eliminate the Major Allergen Gly m Bd 30K by RNA Interference-mediated Gene Silencing

摘要

Gly m Bd 30K has been recognized as a major allergenic protein in soybean (Glycine max (L.) Merr.l seeds, and it exists in nearly all of the soybean cultivars. This study attempted to eliminate the immunodominant Gly m Bd 30K protein from soybeans using RNA interference (RNAi). RESULTS: A 395-bp fragment from the Gly m Bd 30K gene coding region was designed to target the Gly m Bd 30K gene. The 30K-specific RNAi transformation cassette was subcloned into a binary pCAMBIA3301 vector to construct the plasmid pCAMBIA-30K-RNAi. Transgenic soybeans were generated by infecting soybean cotyledonary node explants with Agrobacterium tumefaciens EHA105. Three independent transgenic soybean plants were confirmed by PCR and Southern blot analyses. Quantitative real-time PCR (qRT-PCR) analysis of gene expression in the transgenic seeds indicated that there was trace accumulation of Gly m Bd 30K mRNA. Western blotting demonstrated the absence of Gly m Bd 30K protein in the crude extracts of transgenic seeds. In addition, compared with wild-type plants, the RNAi plants showed no apparent phenotypic and no obvious developmental changes. CONCLUSION: Our study yielded new soybean germplasm with Gly m Bd 30K-null seeds and demonstrated the feasibility of alleviatingsoybean allergies using RNAi technology.