Production of Hypoallergenic Rice by Gene Engineering

一些关于谷物过敏反应的研究表明，一些特应性食物过敏患者的血清中含有与大米蛋白反应的免疫球蛋白E (IgE)，可溶性蛋白、白蛋白和球蛋白具有高度的致敏活性。我们在水稻过敏患者中分离到一个约16kDa的水稻种子蛋白，该蛋白对IgE具有反应性，后来被鉴定为水稻种子中主要的过敏原之一。从成熟水稻种子cDNA文库中分离到一个编码水稻主要致敏蛋白（RAP）的cDNA克隆（RA17）。应用反义RNA策略抑制成熟水稻种子RAP基因的表达。通过电穿孔将CaMV 35S启动子、水稻蛋白启动子和RAP启动子控制的RAP反刍rna嵌合基因引入到水稻中。从水稻悬浮细胞中分离的原质体与每个反肽基因和与CaMV 35S启动子相关的一个潮霉素磷酸转移酶（HPT）基因共转化，水稻植株从抗潮霉素愈伤组织中独立再生。从再生植株叶片中分离总DNA，用限制性内切酶酶切，以RAP cDNA为探针进行Southern blot分析。在几个克隆中，观察到一个具有预期大小的反义基因的额外带。利用RAP特异性单克隆抗体，采用SDS-PAGE/免疫印迹法和ELISA法检测RAP含量。部分转基因植物种子的RAP条带明显弱于野生型对照。对转基因和野生型水稻种子中提取的蛋白质进行ELISA分析，发现RAP含量降低至原始水稻种子的30%左右。引入的反义基因在转化后代中稳定传递和表达的研究正在进行中。

Several studies on allergic reaction to cereal grains have suggested that sera from some patients with atopic food allergy contain immunoglobulin E (IgE) reacting with rice proteins, and that soluble proteins, albumins and globulins have a high degree of allergenic activity. We isolated a rice seed protein of about 16kDa with reactivity for IgE in rice-allergic patients, and the 16kDa-protein was later identified as one of the major allergenic in rice seed.A cDNA clone (RA17) encoding the rice major allergenic protei (RAP) was isolated from cDNA library of maturing rice seeds. An antisence RNA strategy was applied to repress RAP gene expression in maturing rice seeds. Some chimeric genes encoding antisence RNAs of RAP under the control of CaMV 35S promoter, rice prolamin promoter and RAP promoter were introduced into rice by electroporation. Protophasts isolated from rice suspension cells were co-transformed with each antisence gene and a hygromycin phosphotransferase (HPT) gene linked to the CaMV 35S promoter, and rice plants were independently regenerated from hygromysin-resistant calli. Total DNA isolated from leaves of the regenerated plants was digested with some restriction enzymes, and Southern blot analyzes were done using an RAP cDNA as a probe. In several clones, an additional band with an expected size of the antisence gene was observed. Selfed seeds from these transgenic plants with the antisence gene were tested for RAP content by SDS-PAGE/immunoblotting and ELISA using a monoclonal antibody specific for RAP.The RAP bands stained with the antibody of seeds from some transgenic plants were much weaker than those of the wild-type control. ELISA analysis for proteins extracted from the trangenic and wild-type rice seeds suggested that RAP content of rice seeds was reduced to about 30% of the original one. Studies on stable transmission and expression of the introduced antisence gene among the transformant progeny are in progress.