Study On The Mechanism Of Low Nitrogen Tolerance Mediated By Tibetan Wild Barley HvbHLH56

Nitrogen is an essential nutrient element for plant growth,and it is a limiting factor for increasing crop yield in agricultural production.In recent years,excessive use of nitrogen fertilizer has caused environmental pollution problems.Further analysis of the low nitrogen tolerance mechanism of crops plays an important role in reducing the dependence of crops on nitrogen fertilizers and promoting the rapid development of agriculture.In the previous study,a low nitrogen-tolerant candidate gene belonging to the bHLH family was identified through transcriptome analysis of the low nitrogen-tolerant variety XZ149 identified in wild barley.Firstly,members of barley bHLH family were identified through the genome-wide analysis,and then the characters and evolutionary relationships among them were analyzed.The gene was named as HvbHLH56,because it has the highest homology with Os01g72730(OsbHLH 056).Subsequently,the gene was cloned in Tibetan wild barley XZ149,and its response mechanism to low nitrogen was analyzed through its subcellular localization and overexpression in Arabidopsis under low nitrogen stress.The main results of this study are as follows:1.Total of 132 HvbHLH transcription factor members were identified in barley employing bioinformatics methods.Of which,126 were unevenly distributed on 7 chromosomes of barley,and 6 genes were not localized on any chromosomes.Physicochemical analysis showed that proteins encoded by the HvbHLHs consist of 86 to 767 amino acid residues,the maximum molecular weight reached 83538.67 Da,the minimum value was 12014.64 Da,and their isoelectric point pI ranged from 4.63-11.9.Multi-sequence alignment,analysis of gene structure,motif,and phylogenetic tree showed that the barley bHLH family is divided into 24 subfamilies,each of which has a different genetic structure and motif.The number of exons/ introns and the motif structure in the same subfamily were basically the same.GO enrichment analysis sugeested that barley bHLH genes was significantly enriched in molecular biological function,with DNA binding,protein dimerization activity,and protein binding function.2.HvbHLH56 gene was successfully cloned from low nitrogen tolerant wild barely genotype XZ149.A recombinant overexpression vector HvbHLH56:GFP was constructed.Its transient expression in tobacco showed that HvbHLH56 protein was expressed in the nucleus and cell membrane.3.The Arabidopsis with overexpression of HvbHLH56 was obtained by Agrobacteriummediated method,and positive homozygous plants were acquired by means of generations propagation.Under low nitrogen stress,the decrease in fresh weight and lateral root number of transgenic Arabidopsis was significantly lower than that of wild type;meanwhile,the root length of transgenic Arabidopsis was increased or no changed,but the root length of wild type was significantly reduced.It indicated that the,overexpression of HvbHLH56 in Arabidopsis significantly alleviated the inhibition of plant growth by low nitrogen stress.In addition,the decrease of nitrate nitrogen and soluble protein content in transgenic Arabidopsis was significantly lower than that of wild type,while the activities of nitrate reductase,glutamine synthase,and glutamate synthase increased more than wild type under low nitrogen stress.These results indicated that the overexpression of HvbHLH56 in Arabidopsis can may enhance its ability to uptake and assimilate nitrogen in the absence of nitrogen,which may be contribute to the low nitrogen tolerance in transgenic plants.