Functional Analysis Of HvJRL34 In Response To Low Nitrogen Stress In Wild Barley

Nitrogen is an essential nutrient element for plant growth and development.Currently,traditional method to increase crop yield depends on the application of nitrogen fertilizer.However,low use efficiency of nitrogen fertilizer and high amount of nitrogen fertilizer used caused serious environmental pollution and waste of resources.One of the effective ways to reduce nitrogen fertilizer used and increased crop yield is to breed low-nitrogen tolerant varieties through crop genetic modification.Barley is an important grain crop.After long-term breeding and genetic improvement,the genetic base became single.Thus,it is difficult to make a big breakthrough in quality and resistance in barley cultivars.Tibetan wild barley is a valuable germplasm resource for yield breeding and resistance to stress.In the previous study,we have identified a candidate gene belonging to Jacalin lectin(JRL)family involved in low nitrogen tolerance by comparative transcriptome analysis between the tolerant genotype XZ149 and the sensitive genotype XZ56.In the present study,we firstly identified and analyzed the characteristics and evolutionary relationships of these family members at the genome-wide level in barley.Subsequently,the gene was cloned from Tibetan wild barley XZ149,and its structure,spatiotemporal expression characteristics,and function in Arabidopsis were analyzed to preliminarily elucidate the mechanism of its response to low nitrogen.The main results of this study are as follows.1.Total of 35 JRL family members were identified by HMMER search and BLASTP analysis,34 of them were unevenly distributed on seven barley chromosomes.The candidate gene was located on Chromosome 7 and named HvJRL34 according to its chromosomal location.HvJRL family proteins consisted of75-1232 amino acid residues,and their molecular weights varied from 7777.89 to138276.88 Da,with an isoelectric point between 4.08 and 9.64.Through interspecies phylogenetic analysis of barley,wheat,Arabidopsis,and rice,176 JRL proteins were classified into 10 subfamilies.Members of the barley JRL family showed diversity in gene structure,motifs,and structural domains,however the gene structure and motif composition were largely consistent across subfamilies.The tissue expression results indicated that the expression of the family members was tissue-specific.2.Semi-quantitative and q PCR analyses showed that the gene was expressed in roots,stems,and leaves,but the expression was highest in roots.Under low nitrogen stress,the expression of low nitrogen tolerant genotype XZ149 was up-regulated in leaves from 1 h to 12 d and continuously up-regulated in roots for 2 d,while the expression of low nitrogen sensitive genotype XZ56 was down-regulated in leaves after 24 h and up-regulated in roots only at 3 h and 6 h.The continuous high expression of the HvJRL34 gene in XZ149 might contribute to its higher tolerance for low nitrogen.To further verify the result,the HvJRL34 gene was successfully cloned from the low-nitrogen tolerant genotype XZ149,and the HvJRL34: YFP overexpression vector was constructed to infiltrate tobacco.Subcellular localization of tobacco protoplasts showed that the protein was localized in the cytoplasm.3.The transgenic Arabidopsis thaliana with high expression of HvJRL34 was obtained by Agrobacterium-mediated method and the pure-hybrid transgenic Arabidopsis thaliana was generated by addition generation.Under low nitrogen conditions,root length,lateral root number and ground fresh weight of wild type and transgenic Arabidopsis thaliana were inhibited by low N.But under low N stress,root length,lateral root number and fresh weight of transgenic Arabidopsis thaliana were significantly higher than wild type,and their declines were lower than wild type.The results indicated that Arabidopsis with high expression of HvJRL34 had stronger low nitrogen tolerance.Also,the decrease in nitrate-nitrogen content and soluble protein content of transgenic Arabidopsis under low nitrogen conditions was significantly lower than that of the wild type,but the increase in nitrate reductase and glutamine synthetasewas higher than that of the wild type.These results suggested that overexpression of HvJRL34 in Arabidopsis may enhance its nitrogen assimilation ability under low nitrogen stress,which may help to improve low nitrogen tolerance in transgenic plants.