Cloning Of Hulless Barley HvPrx2 Gene And Study On Its Function Under Cold Stress

For plants,the maintenance of intracellular redox homeostasis is very important in normal growth and in response to various environmental stresses.In plants and cyanobacteria,peroxiredoxins（Prxs）are key molecules in redox signaling pathways in plants and cyanobacteria.At present,the research on the structure and function of Prxs protein has gradually attracted widespread attention.Hulless barley（Hordeum vulgare L.var.nudum Hook.f.）is a unique cereal crop in China.It is only distributed in the high-altitude area,such as Qinghai-Tibet Plateau and its adjacent areas.Because of its robust low temperature stress tolerance and adaptability,it is considered as a potential crop cold-resistant germplasm resource and genetic resource pool.Since 2015,with the accomplishment of basic research work such as transcriptome and proteome sequencing under various restricted growth conditions,research on the genetic characteristics and the mechanism of hulless barley of stress resistance has gradually attracted the attention of researchers.Based on the transcriptome data,an orthologous gene of Prxs family BAS1 of cultivated barley was cloned from hulless barley and designated as HvPrx2.Then,different Arabidopsis thaliana transgenic lines overexpressing HvPrx2 were constructed（HvPrx2OE lines）.On this basis,we measured different phenotypes of each transgenic Arabidopsis thaliana lines and the control plants under different temperature treatment conditions from different levels,and preliminarily explored the possible function of HvPrx2 gene in the low temperature stress resistance of plants.The main research results obtained in this work are as follows:1.Transcription data of hulless barley seedlings treated with different time of low temperature stress showed that:under low temperature stress for 16 h,the enrichment and expression of genes involved in the regulating of antioxidant activity in cells were significantly boosted compared with that of them under low temperature stress for 4 h.2.The open reading frame sequence（ORF）of the orthologous gene HvPrx2 of cultivated barley BAS1 was cloned from hulless barley.Although HvPrx2 lacked 102 bases compared with barley BAS1,the protein structure and evolutionary relationship between the two were highly homologous.3.Cell-PLoc prediction showed that HvPrx2 protein may be localized in chloroplast and cytoplasm,and subcellular localization experiments of transient transformation of Arabidopsis thaliana protoplasts confirmed that HvPrx2 protein was localized in chloroplasts.4.The p RI101AN-HvPrx2 binary expression vector was constructed,and the HvPrx2transgenic plants（HvPrx2 OE lines）were obtained and screened in Arabidopsis thaliana of different genetic backgrounds by Agrobacterium-mediated floral-dipgentic transformation method.And the possible functions of HvPrx2 in response to low temperature stress were preliminarily explored in transgenic plants:At 4°C,root and leaf growth of Arabidopsis thaliana seedlings were significantly inhibited,and and the knockout lines of Prx-A and Prx-B genes showed obvious photosynthesis damage;while these inhibitory effects were relatively weaken in the HvPrx2 OE lines.At 4°C,the heterologous transfer HvPrx2 of hulless barley could affect the functions of the key genes At CBF1 and At COR47 of cold-responsive genes and the key genes of the chloroplast thioredoxin system At Trx1 and At NTRC.Meanwhile,it could alleviate the photosynthesis inhibition caused by low temperature stress to some extent.At 4°C,heterologous HvPrx2 could enhance the ability of plants to scavenge intracellular H₂O₂ and maintain intracellular redox under low temperature stress by affecting the activities of key antioxidant enzymes such as POD and CAT and the expression patterns of their encoding genes.In summary,HvPrx2 could coordinate the balance of energy allocation requirements between vegetative growth and stress resistance in plants under low temperature stress by influencing for the functions of some key stress resistance genes and regulating the activities of redox-related enzymes,so as to synergistically achieve the intracellular redox homeostasis and enhancement of stress tolerance of transgenic Arabidopsis thaliana under low temperature stress.It suggested that the maintenance of redox homeostasis by HvPrx2may be the reason for the strong tolerance of hulless barley under low temperature stress.