Effects Of Different Nitrogens On Nitrate Reductase Activity And Expression Of Nitrate Reductase Gene In Soybean

The rate-limited and regulated step in nitrate assimilation appears to be the initial reaction, catalyzed by nitrate reductase(NR). NR plays an important role in plant nitrogen(N) metabolism. Nitrate(NO3-) in the environment is absorbed and used by the high plant through following two assimilation steps: the first step is reduction of NO3- to NO2-, catalyzed by NR. Second, Nitrite reductase reduce NO2- to NH4+. And last, NH4+ is used to synthesize animo acid and protein with relative enzymes. So studying NR activity(NRA) and its application is very important in plant breeding and agricultural development. Nitrate assimilation remains the major pathway of plant use N. In this experiment, effects of different nitrogens(NO3--N,NH4+-N,NO3--NH4+)on NRA, NR gene expression and seed protein contents of soybean cultivars during pod-bearing period were investigated. The experimental results indicated that NR activities in leaves of six soybean genotypes treated by NO3--N were obviously enhanced. However, NR activities of these genotypes treated by NH4+-N were the lowest in three kinds of nitrogens. Seed protein contents of six genotypes induced by three kinds of nitrogens were higher than those done by water which is the control group. The correlation between NR activities and protein contents was extremely remarkable (r= 0.671**). Hence, it is suggested that high NR activity in soybean leaves should be one of bio-chemical index in selecting soybean germplasms with high seed protein.We designed the primers according to the known NR sequence in the GenBank, then cloned the NR cDNA fragment using the method of RT-PCR. The extracted RNA cross-linked with a-32P-labeled NR cDNA probe. Northern blot analysis showed that NR mRNA have the most obviously hybrid signal induced by NO3--N. This proved that accumulations of NR mRNAs in six genotypes induced by NO3--N were much more than those treated by the other two kinds of nitrogens and water. NR gene expressions in six soybean genotypes treated by three kinds of nitrogens were different. The orders of accumulations of NR mRNA were: NO3- > NO3--NH4+> NH4+, which showed that different nitrogen sources promote the expression of NR gene not only on the level of transcription, but also on the level of NR activity. Moreover, amounts of NR mRNAs of three high-protein genotypes were higher than those of three low-protein genotypes treated by the same nitrogen source.