Transcriptome Response Characteristics Of Millet To Different Nitrogen Forms

Nitrogen is the most needed nutrient element in the process of crop growth and development.It mainly exists in the form of ammonium nitrogen and nitrate nitrogen in soil.It is of great significance to explore the mechanism of nitrogen absorption and utilization and how to improve the nitrogen absorption and utilization efficiency of crops for the development of resource-saving and environment-friendly agriculture.Millet is tolerant to barren and has high nitrogen absorption and utilization efficiency.It is an ideal material to study the mechanism of efficient absorption and utilization of nitrogen in crops.In this study,a new type of millet xiaomi was used as research material to study the optimal concentration of ammonium salt and low ammonium salt stress for xiaomi growth.Then,the concentration was used to treat xiaomi with low ammonium salt stress for different times,to screen and verify the key differentially expressed genes related to ammonium salt,and to explore the gene expression regulation mode of xiaomi in response to low ammonium salt stress at transcriptome level.Final,the transcriptome of xiaomi treated with nitrate was compared and analyzed to explore the key differential genes that jointly regulate the absorption and transport of ammonium and nitrate.The aim of this study is to lay a foundation for further study on the expression regulation network of nitrogen uptake and transport related genes in Millet.The main results are as follows:(1)Study on the optimum and low ammonium concentration of xiaomi: under the concentration of0.1 mmol/L,0.2 mmol/L,0.5 mmol/L,1.0 mmol/L,2.0 mmol/L,4.0 mmol/L,6.0 mmol/L and 8.0 mmol/L NH4 Cl,the plant height,root length,seedling fresh weight,root fresh weight,chlorophyll content,seedling dry weight and root dry weight were determined: death occurred at 3 days after 0.1 mmol/L treatment;compared with 1.0 mmol/L,2.0 mmol/L,4.0 mmol/L,6.0 mmol/L and 8.0 mmol/L,there were significant differences in 7 indexes under 0.2 mmol/L treatment;compared with 0.5 mmol/L,there were significant differences in plant height,seedling fresh weight,root fresh weight,chlorophyll content and seedling dry weight,it indicated that the stress of xiaomi was the most stress at the concentration of 0.2 mmol/L;under the concentration of 2.0 mmol/L 4.0 mmol/L 6.0 mmol/L and 8.0 mmol/L,there was no significant difference in 7 indexes.Therefore,0.2 mmol/L NH4 Cl was selected as the low ammonium stress concentration of xiaomi,and 2.0 mmol/L was selected as the normal concentration.(2)Transcriptome analysis of xiaomi under low ammonium stress: the transcriptome analysis of xiaomi under 0.2 mmol/L NH4 Cl stress showed that there were 450 genes: 111 differentially expressed genes in the shoot,of which 55.86% were up regulated and 44.14% were down regulated;339 differentially expressed genes in the underground,of which 12.68% were up regulated and 87.31% were down regulated.Through annotation analysis: 13 differential candidate genes related to ammonium uptake and transport were screened in the shoot of which 2 were up regulated and 11 were down regulated;10 were screened in the underground,of which 9 were up regulated and 1 was down regulated.(3)Expression patterns of key candidate genes under low ammonium salt stress: real-time PCR was used to verify the expression pattern of some candidate genes related to ammonium uptake and transport.The results were highly correlated with the results of transcriptome sequencing(R2=0.7278).Under 0.2mmol/L NH4 Cl stress: the relative expression of Si5g20720 was up regulated not only in roots,but also in the shoot,it is supposed to be the key gene responsible for ammonium absorption and transport,the relative expression levels of Si1g11940,Si5g29250,Si1g11910,Si1g24430 and Si9g02830 in roots were up regulated,which suggested that they were involved in ammonium absorption.(4)Comparative analysis of transcriptome under ammonium and nitrate treatment: through transcriptome analysis of xiaomi treated with ammonium and nitrate,2238 genes were screened out in the shoot,of which 37.89% were up regulated genes and 62.11% were down regulated genes;2736 genes were screened out in the underground,of which 64.18% were up regulated genes and 35.82% were down regulated genes.Through analysis,five candidate genes,Si1g11940,Si1g11910,Si5g29250,Si6g17980 and Si1g22620,were screened out.Real-time PCR showed that the relative expression of Si5g29250 was up regulated under the treatment of ammonium and nitrate respectively,which suggested that Si5g29250 might be the key gene of ammonium and nitrate transport;Si6g17980 is the key gene for nitrate transport and ammonium transport;the relative expression levels of Si1g11910 were up regulated in both shoot and underground parts,suggesting that Si1g11910 is a key gene regulating the absorption and transportation of ammonium and nitrate.