Molecular Cloning And Expression Analyses Of Genes Related To Nitrogen Metabolism From Mulberry

Nitrogen nutrient is a necessary element for plant growth and development, and it is the life element of plant. The main nitrogen sources for plants absorption and utilization are ammonium nitrogen and nitrate nitrogen. In order to adapt to different nitrogen environment, plants evolve ammonium transporter and nitrate transporter proteins to classify absorption, translocation and nitrogen metabolism among different nitrogen. The history of Chinese sericulture industry is long. In order to improve the yield of mulberry, reasonable application of nitrogen fertilizer for mulberry is necessary. However, if nitrogen fertilizer is excessive, it will not only cause waste because of non-absorption of mulberry trees, but also cause environmental pollution. Study of mulberry nitrogen metabolism from the angle of molecular biology, is conducive to provide theoretical basis to breed new mulberry variety of high efficient nitrogen absorption ratio and improve the nitrogen absorption efficiency of mulberry, and it will be significant for mulberry breeding.The plant materials are Lu sang Yu 71-1 seedlings in this experiment. According to the specific primers which were designed according to the homology analyses of each species, we cloned three genes which obtained complete open reading frame by RT-PCR, rapid amplification of c DNA ends(race) and software mosaic technology, and carry out a series of bioinformatics analysis. Expression analyses of three genes cloned by using different nitrogen treatments and fluorescence quantitative PCR. The main research contents are as follows: 1. Cloning, sequence analysis and expression analyses of AMT1 gene of mulberryThe Mm AMT1 gene, which contains a complete open reading frame, was cloned from Lu sang Yu 71-1. The length of the sequence was 1518 bp, encoding 505 amino acid residues, which belonged to Transporter Family Ammonium. The homology between different plant species was analyzed by bioinformatics. Under different nitrogen conditions, real-time PCR results show that in treatments of different concentration of KNO3, expressions of Mm AMT1 gene were increased volatility at low and medium concentration NO3- treatments, but decreased volatility at high concentration NO3- treatment. Under treatments of different concentration of(NH4)2SO4, the expression of Mm AMT1 gene was increased under low concentration NH4+, but decreased under medium and high concentration NH4+. Under treatments of different nitrogen sources, the expression of Mm AMT1 gene was merely increased under NH4NO3 treatment. 2. Cloning, sequence analysis and expression analyses of AMT2 gene in MulberryMm AMT2(Application patent No.: 201610203308.5) was cloned from Lu sang Yu 71-1. It’s complete open reading frame sequence 1470 bp long, encoding 489 amino acid residues, belonging to the ammonium transporter family. The homology between different plant species was analyzed by bioinformatics. In different nitrogen conditions, real-time PCR results showed that Mm AMT2 gene has different levels of response and expression changes. In treatments of different concentration of KNO3, the general trend of expression quantity of Mm AMT2 gene was increased firstly until the maximum, then decreased, and the responsivity of low concentration NO3- was lower than that of medium concentration and high concentration NO3-. Under treatments of different concentration of(NH4)2SO4, the expression of Mm AMT2 gene was increased under low concentration NH4+, and decreased under medium and high concentration NH4+. Under treatments of different nitrogen sources, the expression of Mm AMT2 gene was only increased under KNO3 treatment. 3. Cloning, sequence analysis and expression analyses of NRT1 gene of mulberryMm NRT1(Application patent No.: 201610203634.6)was cloned from Lu sang Yu 71-1. The sequence is 1062 bp long, its open reading frame sequence is 819 bp long, encoding 272 amino acid residues, belonging to the MFS super family. Analyze its homologic among different plant species by bioinformatics. Under different nitrogen conditions, real-time PCR results showed that in treatments of different concentration of KNO3, the overall trend of expression quantity of Mm NRT1 gene was increased firstly, and then decreased. The responsivity of low concentration NO3- was lower than that of medium concentration and high concentration NO3-. Under treatments of different concentration of(NH4)2SO4, the expression of Mm NRT1 gene was increased firstly and then decreased, and with little change under low concentration NH4+. Under medium and high concentration NH4+, the expression of Mm NRT1 was increased to a maximum value, followed by a slight reduction, the overall expression was increased. Under treatments of different nitrogen sources, the expression of Mm NRT1 gene was only increased under NH4NO3 treatment.