Screening And Identification Of Nitrate Regulatory Mutant In Arabidopsis

Nitrogen is one of a large number of essential macronutrients for plant growth and development. It involved in the synthesis of proteins, chloroplast, nucleic acids, coenzyme, enzyme and various nitrogenous substances. It is widely distributed in various tissues and organs of plants. The widespread application of nitrogen fertilizer increased global agricultural production. In recent years, however, Nitrogen fertilizer use efficiency of crops reduced year by year. Nitrogen fertilizer cannot be effectively used by crops, and the surplus nitrogen which flew into the environment would cause the problems of environment pollution. It's critical to strengthen the study of nitrogen, and to cultivate the high-nitrogen-utilized efficiency variety as well as to improve the nitrogen uptake of crop, in order to guarantee the high yield and the sustainable development of agriculture.Nitrate is the main-utilized nitrogen form for plants, it absorbed by the high-/low-affinity transport systems from the soil, then transported to roots or leaves, and assimilation to amino acid by nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthetase finally. It involved in the synthesis of living matters such as protein. Nitrate is not only a nutrient, but also a biological signal. Plants can regulate life activities just like root development via response to nitrate signal, to adapt to complex environment. Nitrate-regulatory genes play an important role in this response process, and clearing the nitrate regulatory gene network is the key to solving the problem. In recent years, the focus of nitrogen research have turn from nitrate transporter to nitrate regulator, and so far several nitrate regulatory genes have been found, such as NRT1.1, NLP7, CIPK8/23, LBD37/38/39, NRG2 and microRNA167/393.In this study, we screened and identified a nitrate regulatory mutant by the above mutant screening system:First of all, we mutagenized the screening system SS204-9 by ethyl methane sulfonate. Then we screened one mutant Mut136 by inverted fluorescence microscope from the M2 generation of segregation population, which grew in ammonium nitrate.Secondly, we positioned the mutated site of Mut136 in the front of chromosome IV(10.29M-10.89M) by map-based cloning as well as high-throughput sequencing, and determine two candidate mutated genes to be AN1 and AN2.Thirdly, we disposed Mut136 and WT with KNO3, and then measured its NO3- response genes in the body expression(NIA1, NiR, NRT2.1). The expression of Mut136 is lower than in the wild type. It shows that Mut136 is NO3- regulatory mutant.Fourth, we cultivated Mut136 and WT under the condition of different concentrations of KNO3, statistics and main root length, lateral root number and root density. We can find that in low or relatively high concentrations, and Mut136 there exist significant difference with WT. Thus it can be seen, the mutated gene may be associated with root system built or ion stress.Fifth, we determined the nitrate nitrogen content, nitrate reductase activity, amino acid content of Mut136. Nitrate nitrogen content in root of Mut136 is higher than that in WT, while the NR activity and amino acid content is lower than WT. These results support that the mutated gene of Mut136 involved in the nitrate nitrogen metabolism.