NITRATE REDUCTASE FROM SQUASH - CDNA CLONING AND NITRATE REGULATION
NITRATE REDUCTASE FROM SQUASH - CDNA CLONING AND NITRATE REGULATION
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DOI：
10.1073/pnas.83.21.8073
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发表时间：
1986-11-01
影响因子：
11.1
通讯作者：
DAVIS, RW
中科院分区：
文献类型：
--
作者：
CRAWFORD, NM;CAMPBELL, WH;DAVIS, RW
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，一种硝酸诱导酶。硝酸处理对西葫芦幼苗子叶的硝酸还原酶活性和蛋白质均有诱导作用。从硝酸盐处理过的幼苗子叶中制备Poly(A)+ RNA，并利用其构建。gt11 cDNA文库。利用小鼠的抗体免疫纯化的南瓜硝酸还原酶，重组。分离到编码部分硝酸还原酶mRNA的噬菌体。利用重组噬菌体产生的抗原亲和纯化免疫小鼠腹水的抗硝酸还原酶抗体。纯化后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。在硝酸盐诱导的子叶中，插入的cDNA（1.2千碱基）与3.2千碱基的RNA杂交，其丰度是未诱导组织的120倍。
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 .lambda.gt11 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.