Nitrate reductase from squash: cDNA cloning and nitrate regulation

Area of Science:

• Plant molecular biology
• Biochemistry
• Enzymology

Background:

• Nitrate assimilation is crucial for plant growth, involving nitrate reductase (NR).
• NR is a nitrate-inducible enzyme, but its molecular regulation in plants is not fully understood.
• Understanding NR induction is key to optimizing plant nitrogen use efficiency.

Purpose of the Study:

• To isolate and characterize the complementary DNA (cDNA) encoding nitrate reductase from squash (Cucurbita maxima L.).
• To investigate the molecular basis of nitrate-inducible NR expression in plant cotyledons.

Main Methods:

• Construction of a lambda gt11 cDNA library from nitrate-induced squash cotyledon poly(A)(+) RNA.
• Screening the library using antibodies against purified squash NR.
• Characterization of the isolated cDNA insert and its corresponding RNA transcript.

Main Results:

• A recombinant lambda phage encoding a portion of squash NR mRNA was isolated.
• The isolated cDNA insert (1.2 kb) hybridized to a 3.2 kb RNA transcript.
• This transcript was 120-fold more abundant in nitrate-induced cotyledons than in uninduced ones.
• The purified antibody specifically recognized and immunoprecipitated squash NR.

Conclusions:

• Nitrate induces the expression of NR at the mRNA level in squash cotyledons.
• The isolated cDNA provides a tool for further studying NR gene regulation in plants.
• This research contributes to understanding the molecular mechanisms of nitrate assimilation in plants.