Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 1986 Nov;83(21):8073-6. doi: 10.1073/pnas.83.21.8073.
The assimilation of nitrate in plants involves the reduction of nitrate to ammonia in two steps. The first step requires nitrate reductase, a nitrate-inducible enzyme. When seedlings of squash (Cucurbita maxima L.) were treated with nitrate, both nitrate reductase activity and protein were induced in the cotyledons. Poly(A)(+) RNA was prepared from cotyledons of nitrate-treated seedlings and was used to construct a lambdagt11 cDNA library. Using antibodies from mice immunized against purified nitrate reductase from squash, a recombinant lambda phage was isolated that encoded part of the nitrate reductase mRNA. The antigens produced by the recombinant phage were used to affinity purify anti-nitrate reductase antibody from ascites fluid of immunized mice. The purified antibody bound to nitrate reductase protein on immunoblots and immunoprecipitated the enzyme from squash protein extracts. The cDNA insert (1.2 kilobases) hybridized to a 3.2-kilobase RNA that was 120-fold more abundant in nitrate-induced cotyledons compared with the uninduced tissue.
植物对硝酸盐的同化包括将硝酸盐还原为氨的两个步骤。第一步需要硝酸还原酶，这是一种硝酸盐诱导酶。当南瓜（Cucurbita maxima L.）的幼苗用硝酸盐处理时，子叶中的硝酸还原酶活性和蛋白都被诱导。从硝酸盐处理的幼苗的子叶中制备聚（A）（+）RNA，并用于构建 lambdagt11 cDNA 文库。使用从小白菜中纯化的硝酸还原酶免疫小鼠产生的抗体，从重组λ噬菌体中分离出编码部分硝酸还原酶 mRNA 的噬菌体。重组噬菌体产生的抗原用于从免疫小鼠的腹水液中亲和纯化抗硝酸还原酶抗体。纯化的抗体在免疫印迹上与硝酸还原酶蛋白结合，并从南瓜蛋白提取物中免疫沉淀该酶。cDNA 插入片段（1.2kb）与在硝酸盐诱导的子叶中比未诱导的组织丰富 120 倍的 3.2kb RNA 杂交。
