Cloning And Functional Analysis Of NAC Transcription Factors Associated With Barley Yellow Mosaic Disease

Barley is an important cereal crop in the world.It has a wide area for planting and has a long history of planting in China.It is mainly used as feed for livestock,raw materials for food processing,and brewing beer.Barley yellow mosaic virus is a soil-borne virus.The virus is parasitic in the spores of Aspergillus gracilis in the soil.It has the characteristics of wide incidence,rapid propagation,and high infection rate.It causes the loss of even more than half of the winter barley varieties.Transcription factors play an important role in plants’ resistance to external stress.In this study,NAC transcription factors in barley were studied to search for the NAC gene related to yellow mosaic disease and to study its function.In the present study,the susceptible materials of Barley yellow mosaic disease,Morex,Igri,and Barke,were used to identify the regulatory networks involved in the pathogenesis of Barley yellow mosaic disease through transcriptome sequencing.The two genes related to barley yellow mosaic disease were obtained through the technique of pathway cloning.A NAC transcription factor,which performs bioinformatics analysis to predict the expression characteristics of the gene and its evolutionary relationship with other species;Agrobacterium-mediated subcellular localization after vector construction;Yeast double-hybrid technique to study how transcription factors interact with proteins.Understanding the roles of two NAC transcription factors HvNAC6(HORVU5Hr1G011650)and HvNAC7(HORVU7Hr1G106480)in barley-infected BaMMV provided the basis for the later development of anti-barley yellow mosaic disease varieties.1.According to the result of RNA-seq analysis,out of a total of 37,804 genes in this transcript,10,164 genes were effectively differentially expressed after infection of Igri seedlings with M.oleracea,of which 5,192 were up-regulated,4,972 were down-regulated.Among them,534 transcription factors were predicted and belong to different families of transcription factors.2.Among the predicted transcription factors,there were two significant up-regulation,HvNAC6 and HvNAC7.Through tissue expression analysis,it was found that HvNAC6 was significantly expressed in both the late fruiting stage and the outer glume,but it was not expressed in the young panicle and there was a weak expression in the newborn ear.HvNAC7 was significantly expressed in the young panicles.As analyzed by conserved domains,both NAC transcription factors have a NAC family conserved domain.3.The subcellular localization of HvNAC6 and HvNAC7 by Agrobacterium tumefaciens-mediated tobacco infection showed that both of them are nuclear localization proteins and perform corresponding functions in the nucleus.4.Transcription factors in the yeast system were constructed by yeast two-hybridization with the BaMMV viral protein construct,the susceptibility factor eIF4 E,and the recessive resistance gene PDIL5-1.Protein interaction was not found.This indicates that it does not directly affect the process of infecting BaMMV and may be more of a regulatory or modified transcription factor in this process.5.Different expression of HvNAC6 and HvNAC7 in resistant materials was analyzed by qRT-pcr technology.Only the expression level of HvNAC7 in 24 HLB disease sensitive materials was found to be significantly higher than that in 24 HMD-resistant materials,indicating that HvNAC7 has an important relationship with the regulation of response to infection with barley yellow mosaic disease.