Function Of MdDREB2A And Its Downstream Target Genes In Response To Apple Alternaria Blotch

China is the largest apple producer with the largest planting area all over the world.Currently,apple Alternaria blotch widely occur in major production areas,severely affecting the industry’s sustainable development.Exploring genes which related to the response to apple Alternaria blotch is crucial to fully utilize its genetic resources and enhance disease resistance.Md DREB2 A is a dehydration-responsive element-binding protein.In this study,RT-q PCR and detached leaves inoculation experiments were used to investigated the function of Md DREB2 A under infecting of Alternaria alternata f.sp.mali(A.alternata).Three downstream target genes of Md DREB2 A,Md PR10s(Md PR10.1,Md PR10.2,Md PR10.3),were identified through Ch IP-q PCR and EMSA.Then,sequence analysis and RT-q PCR were used to study the sequence characteristics and expression patterns of Md PR10 s.Moreover,the study also explored the functions of Md PR10 s under A.alternata infection in overexpressing transgenic apples and investigated the effect of Md PR10.1,Md PR10.2,and Md PR10.3 on the growth of A.alternata through protein prokaryotic expression.The main results are as follows:1.The expression and protein accumulation of Md DREB2 A decreased with the prolongation of infection time by A.alternata.After inoculating A.alternata on detached leaves,the lesion area of Md DREB2 A overexpressing transgenic lines were significantly higher than GL-3,while the lesion area of Md DREB2 A RNAi lines were smaller than that of GL-3,indicating that Md DREB2 A negatively regulates apple resistance to apple Alternaria blotch.Three downstream target genes of Md DREB2 A were identified,which encoded pathogenesis-related(PR)proteins containing CCGAC binding elements in their gene promoters,named Md PR10.1,Md PR10.2 and Md PR10.3.Before and after infection by A.alternata,compared with GL-3,RT-q PCR results showed that m RNA levels of Md PR10 s were lower in Md DREB2 A overexpressing transgenic plants,while higher in Md DREB2 A RNAi plants.Ch IP-q PCR analysis validated the downstream target genes of Md DREB2 A,and further EMSA experiments confirmed that Md DREB2 A could directly bind to the CCGAC motif in the promoters of Md PR10.1,Md PR10.2 and Md PR10.3.2.The amino acid sequences of Md PR10.1,Md PR10.2 and Md PR10.3 were analyzed.It was found that they had high similarity and conserved Bet v 1 domain,suggesting that they may have similar functions.Then,RT-q PCR results revealed that the expression of Md PR10 s was induced by A.alternata infection and expressed in almost all growth stages of apple.The Md PR10s-p K7203 vectors were constructed,and Md PR10.1,Md PR10.2 and Md PR10.3overexpressing transgenic apples were obtained by an agrobacterium-mediated transient transformation system.After inoculating detached leaves,the lesion area of Md PR10s overexpressing plants was significantly lower than GL-3.These results indicate that Md PR10.1,Md PR10.2 and Md PR10.3 positively regulate apple resistance to apple Alternaria blotch.In addition,in vitro experiment showed that the mycelial growth areas of A.alternata were significantly smaller on PDA medium containing Md PR10s proteins than PDA medium with MBP protein or 50m M Tris-HCl(p H=8.0),suggesting that Md PR10.1,Md PR10.2 and Md PR10.3 can directly inhibit the growth of A.alternata and Md PR10s proteins have antifungal activity.