The Molecular Mechanisms Of Cold-tolerance Or Brown-spot Resistance Related Genes In Tobacco

Tobacco(Nicotiana tabacum L.)is an important research model plant,and an important economic crop in the world.In recent years,cold temperature and brown spots are two of the most destructive plant diseases of tobacco production.Transgenic modifying of tobacco to improve its agronomic performance and stress-tolerance is of great importance for promoting tobacco quality and production in the world.In view of the tobacco cold-tolerance works,Compared to CB-1,K326 has superior cold tolerance.It is usually regarded as a germplasm resource with superior cold-tolerance,previous comparative transcriptome analysis for K326 and CB-1 identified several cold-tolerance candidate gens.A low-temperature response bHLH transcription factor NtbHLH123 was cloned and characterized.On the basis of genetic transformation system,the function of NtbHLH123 on the improvement of tobacco cold tolerance was studied.In view of tobacco brown spot disease,transcriptome analysis of different resistance tobacco cultivars,provided new strategies for finding key genes and signal transduction pathways.In this study,we characterized key genes of hypersensitive-induced response genes NtHIRs,and Y2H assay showed that NtHIRs can form homo-or hetero-dimer and can also interact with NtLRR/NtSYP/NtWRKY and so on.Genetic transformation and virus vector-induced gene silencing system were used to further reveal the resistance mechanism of tobacco brown spot disease of interactions between NtHIRs and NtLRR/NtSYP/NtWRKY protein.The main results were as follows:"(1)We characterized a bHLH transcription factor from Nicotiana tabacum,NtbHLH123,in response to cold stress(4?).Overexpression of NtbHLH123 enhanced cold tolerance in transgenic tobacco plants.Based on yeast one-hybrid,chromatin immunoprecipitation PCR,and transient expression analysis assays,NtbHLH123 binds directly to the G-box/E-box motifs in the promoter of the NtCBF genes and positively regulates their expression.Furthermore,NtbHLHl 23-overexpressing plants showed lower electrolyte leakage,reduced malondialdehyde contents,H2O2 and ROS accumulation under cold stress,which contributed to alleviating oxidative damage to the cell membrane after cold stress treatment.And NtbHLH123 increased stress tolerance by improving the expression of a number of abiotic stress-responsive genes to mediate the reactive oxygen species(ROS)scavenging ability and other stress tolerance pathways.(2)The results of phenotype,trypan blue staining and DAB staining showed that,reactive oxygen species were rapidly generated and accumulated in the infested parts of disease-resistant cultivars compared with susceptible cultivars,which promoted cell death and formed an allergic reaction.Meanwhile,the inoculation of Alternaria alternata with different resistance to tobacco varieties,RNA-seq and proteomics analysis showed that the expression of NtHIR2 gene was significantly different.Therefore,an allergic response-inducing gene NtHIR2 was cloned from tobacco and expression analysis showed that NtHIR2 was highly expressed in leaves and was induced by Alternaria alternata invasion and active oxygen induction.The yeast two-hybrid screening library was constructed using NtHIR2 as bait protein.The results showed that NtHIR2 interacts with NtLRR/NtSYP/NtWRKY proteins and may jointly regulate the expression of downstream genes,which may affect plant resistance.