The Morphological And Physiological Characteristics Of Sorghum Under Low Phosphorus And Nitrogen, And The Study Of Sorghum MicroRNA Under Low Nitrogen Stress

Phosphorus and nitrogen are essential elements required for plant growth and development. They are constituents of numerous important compounds including proteins, nucleic acids, amino acids and Chlorophyll. The deficiency of available phosphorus and nitrogen in soils is an important limiting factor for crop yield increase. In agricultural production, abundant nitrogenous and phosphorus fertilizer are used in order to give high and stable crop yields, which often result in low nitrogen and phosphorus use efficiency and leads to serious environmental pollution problems. Therefore, identifying crop germplasm resources for high efficiency utilization of phosphorus and nitrogen, revealing the molecular regulation mechanism of crop’s tolerance to low phosphorus and nitrogenous, and breeding for phosphorus and nitrogen high-efficient crop varieties are essential for the sustainable agriculture. Sorghum is one of a main cereal crop of many countries, which is tolerant to drought, flood and poor soil. In the present study, we investigated the effects of low P and N stress on the morphological and physiological indices of sorghum seedlings, and identified the miRNAs of sorghum under low nitrogen treatment by high-throughput sequencing, verified the expression of candidate miRNAs and their target genes by northern blot in sorghum, and obtained following results:1. Effects of various P levels on sorghum seedlings were investigated by vermiculite culture in pots, the result shows different P supply levels significantly affect the morphologies of sorghum seedlings, but the physiologies of sorghum seedlings were not significantly affected, and P levels of 0.25 mmol/L and 1 mmol/L were found to be the proper levels for low P and normal P treatments; seedling dry weight and P content in shoots were recommended as screening indices for low P-tolerance cultivars at sorghum seedling stage; Bayueqi was identified as a low-P tolerant cultivar, while Nong 858 was a low-P sensitive cultivar.2. Effects of low N stress on the morphological and physiological indices of sorghum seedlings were investigated by hydroponics, the differences of many indices were significant or highly significant between sorghum seedlings under 4 mmol/L and 0.04 mmol/L N levels, the indices included seedling height, stem diameter, plant dry weight, chlorophyll and soluble protein contents. Therefore, the low and normal nitrogen treatment levels were determined as 0.04 mmol/L and 4 mmol/L in sorghum seedling N-stress test, respectively.3. One hundred and twenty-two known miRNAs were obtained in low N-stressed sorghum with high-throughput sequencing, among them the expression differences of 107 miRNAs were observed significant or highly significant between the normal and low N treatments, which belonged to 27 miRNA families., Sixty different expression miRNAs were found in root under the stress of 8 h low N,38 of them were up-regulated, and 22 were down-regulated; when sorghum roots were stressed under 15d low N,22 miRNAs of 41 different expression miRNAs were up-regulated, and 19 were down-regulated; in sorghum leaves,82 differently-expressed miRNAs were found under the stress of 8 h low N,20 of them were up-regulated, and 62 were down-regulated; while 76 differently-expressed miRNAs were found after 15d of low N stress,26 of them were up-regulated, and 50 were down-regulated.4. Target genes of low nitrogen response miRNAs were predicted by bioinformatics approaches,101 target genes were related to 78 differently-expressed miRNAs, which play more than 30 molecular functions, including 55% binding,40% unknown,8% transcription factor activity,7% hydrolase activity, and protein dimerization activity, oxidoreductase activity; signal transducer activity; ATPase activity,etc.5. One hundred and twenty-three novel miRNAs were obtained in sorghum stressed by low N with high-throughput sequencing. In roots,16 differently-expressed miRNAs were found under the stress of 8 h low N,4 were found under the stress of 15d of low N; while in leaves,27 differently-expressed miRNAs were obtained under the stress of 8 h low N,20 were got under the stress of 15d low N. Under the stress of 8 h low N,5 miRNAs were up-regulated in roots and 11 were down-regulated; 4 miRNAs were up-regulated in leaves and 23 were down-regulated; while under the stress of 15d low N,2 miRNAs were up-regulated in roots and 2 were down-regulated,7 miRNAs were up-regulated in leaves and 13 were down-regulated.6. Eight target genes of 4 novel differently-expressed miRNAs were predicted. The molecular functions of the target genes are mainly binding that is DNA binding, protein binding, nucleotide binding; iron ion binding, metal ion binding and pyridoxal phosphate binding, etc.7. Reference genes were screened by Real-time PCR and geNorm. We found that GAPDH and EIF4a were suitable as reference genes of sorghum leaves in Real-time PCR, while sbGAPDH and EIF4a for roots.8. The expression of candidate miRNAs and their target genes in sorghum were verified by Real-time PCR. The similar expression tendencies of these miRNAs were observed with the sequencing result, and the expression levels between most miRNAs and their target genes were negatively correlated.