Screening And Functional Verification Of Nitrogen Utilization Related Genes In Ramie

Ramie is the source of protein feed because of its high protein content and reasonable nutritional structure.The biological yield of ramie is high,and its growth rate is rapid.However,the demand of nitrogen is high and the use efficiency of nitrogen is low,which leads to waste of food resources and serious environmental pollution.Facing with the dual challenges of the development of animal husbandry and the ecological environment in south China,how to improve the nitrogen use efficiency of ramie,and reduce the application amount of nitrogen fertilizer is a problem that we urgently need to solve.Therefore,in this research,"Zhongzhu 1" was used as the research material to study the nitrogen utilization characteristics of ramie under different nitrogen concentrations treatments by the hydroponic experiment.We analyzed the expression pattern of the BnDof genes in ramie under different nitrogen concentrations treatments,and selected the BnDof genes that involved in nitrogen metabolism of ramie.Using transgenic technology,we analyzed that BnGS1 and BnDof14 genes how to affect nitrogen use efficiency in tobacco.The results can be used to understand the nitrogen utilization characteristics of ramie and provide clues for developing new ramie varieties which have high nitrogen use efficiency.The main research results were as follows:(1)Using hydroponic culture experiment,the agronomic characters and nitrogen content of "Zhong zhu1" were analyzed under different nitrogen concentrations: N0(0 mM),N4(4 mM),N8(8 mM)and N12(12 mM).For the growth of plant height,it was the largest under N8,the second was under N12,N4 followed and the smallest under N0.This exhibited that "Zhongzhu1" grown fastest under N8 and slowest under N0.For fresh weight and dry weight of ramie,they reached maximum under N4,followed by N8,and they reached minimum under N0.For the nitrogen content of ramie leaves,it was the highest under N12,followed by N8,N4,it was similar under N8 and N12,the smallest under N0 and it reached significantly different between N4 and N12,N8 and N0.Thought of the growth rate,fresh weight,dry weight and nitrogen content of the plants,we found that "Zhongzhu 1" grown better under N8 condition.Therefore,we speculated that the nitrogen concentration of 8mM might be the optimal concentration for the growth and development of ramie in hydroponics.(2)19 BnDof genes were selected from the genome and transcriptome data of ramie by bioinformatics methods.Phylogenetic analysis showed that BnDof genes were more closely related to MtDof among Dof genes of ramie,Arabidopsis,maize,rice,M.truncatula.Expression patterns of BnDof genes in different tissues of ramie were analyzed.The results revealed that the expression levels of BnDof08 and BnDof12 were higher in flowers,BnDof02,BnDof17 and BnDof18 were expressed higher in leaves,BnDof07,BnDof10 and BnDof19 were expressed higher in stem.This indicated that the expression of BnDof genes reveled spatial specificity.Under the treatments of different nitrogen concentrations,the expression of BnDof gene in ramie root,stem and leaves was analyzed.We found that the expressions of BnDof01,BnDof06,BnDof09,BnDof14,BnDof15,BnDof18 and BnDof19 were correlated with the nitrogen concentration,which was the BnDof genes that may participate in nitrogenutilization of ramie.Analyzing the relationship among Dof genes that involved in nitrogen metabolism in other species and these seven BnDof genes,we found that BnDof01,BnDof07,BnDof14 and BnDof18 deserved more attention.(3)Using genetic engineering methods,overexpression vectors of BnDof14 and BnGS1 were successfully constructed.Through genetic transformation experiments and using qRT-PCR,GUS staining and other multiple validation,BnGS1 tobacco overexpression plants were obtained.The results showed that leaves' nitrogen content and crude protein content of BnGS1 overexpressed tobacco plants was 4.88% and 30.52%,respectively,which were significantly higher than that of wild type tobacco(2.29% and 14.32%).Therefore,it was speculated that BnGS1 gene could improve the nitrogen use efficiency of ramie and could be used as a candidate gene to improve nitrogen use efficiency of ramie.