Study On The Function Of Malus Hupehensis Mh-mi R393a In Response To Apple Alternaria Blotch

Apple is the main cultivar of fruit trees in my country,but fungal diseases severely restrict the development of the apple industry.Malus hupehensis has the characteristics of good affinity,early fruit,high yield,strong stress resistance,etc.,and also has strong resistance to pathogenic bacteria,it is an excellent rootstock for apples.Apple alternaria blotch is a disease caused by fungus,it is a common occurrence in my country’s apple producing areas and is one of the serious fungal diseases that endanger my country’s apple production.Through specific base pairing with target m RNA,mi RNA can cut and degrade or inhibit translation of its target m RNA,thereby exerting a negative regulatory function after transcription.Under pathogen infection,mi RNA can not only regulate the expression of target genes in plants,but also regulate expression of pathogen genes.Studies have shown that mi R393 is involved in the defense of bacterial and fungal diseases,but there is no report about mi R393 in the defense process of woody plant pathogens.In this study,Malus hupehensis was used as the experimental material,and the Agrobacterium-mediated transient transformation of Nicotiana benthamiana was used to compare the promoter activity of Malus hupehensis MIR393a/b/c;The function of MIR393a promoter was analyzed and verified,and ps RNATarget was used to predict Mh-mi R393a target gene,and used 5’RLM-RACE and Agrobacterium-mediated tobacco co-transformation method for mutual verification,RT-q PCR was used to detect the expression patterns of Mh-mi R393a,pre-mi R393a and their target genes Mh TIR1 and Mh AFB2 under the infection of apple alternaria blotch;Gala leaves wild-type WT and Vector as controls,the35SCa MV::Mh MIR393a and 35SCa MV::STTM393a recombinant vectors were transiently transformed,then inoculated with the pathogen of apple alternaria blotch,evaluated the susceptibility,and to explore the mechanism of action of Mh-mi R393a in the infection of apple alternaria blotch.The main findings are as follows:1.Nicotiana benthamiana transiently transformed p MIR393a::GUS and p MIR393b/c::GUS recombinant vectors,histochemical GUS staining and GUS activity quantitative results showed that the MIR393a promoter activity was higher than the MIR393b/c promoter.Malus hupehensis was used as the test material to inoculate the apple alternaria blotch bacteria,the expression levels of mature Mh-mi R393a and precursor pre-mi R393a were analyzed by RT-q PCR at different infection time,the results showed that both were induced by apple alternaria blotch infection.Plant CARE analyzed the cis-acting elements of the MIR393a promoter,and found that the MIR393a promoter contains a large number of cis-elements related to methyl jasmonate（Me JA）and pathogens,and both Me JA and apple alternaria blotch treatments can activate the Mh-mi R393a promoter.2.Predicted the possible target genes of Mh-mi R393a by ps RNATarget,used5’RLM-RACE and Agrobacterium-mediated tobacco co-transformation test for mutual verification,and used RT-q PCR to detect the expression of target genes at different infection times of apple alternaria blotch.The results showed that the candidate target genes of Mh-mi R393a were Mh TIR1 and Mh AFB2;5’RLM-RACE found that Mh-mi R393a acts on the target genes Mh TIR1 and Mh AFB2 by direct shearing,and the cleavage sites all occured between the 10th to 11th bases of the target site of Mh-mi R393a;Histochemical staining of GUS expression and quantification of GUS activity in co-transformed tobacco leaves showed that Mh-mi R393a inhibited the expression of GUS gene in35SCa MV::Mh TIR1-GUS and 35SCa MV::Mh AFB2-GUS through base complementary pairing;the target genes Mh TIR1 and Mh AFB2 were both in response to the infection of apple alternaria blotch,and the relative expression levels of mature Mh-mi R393a and target genes Mh TIR1 and Mh AFB2 showed a certain negative correlation under the infection of the pathogen.3.Overexpression of Mh-mi R393a and inhibition of endogenous Mh-mi R393a in Gala leaves by Agrobacterium tumefaciens-mediated transient transformation,RT-q PCR results showed that transient transformation can effectively regulate the expression of Mh-mi R393a and target genes Mh TIR1 and Mh AFB2.The susceptibility was evaluated after inoculation with the pathogen of apple alternaria blotch,compared with the wild-type WT control and the empty PBI121 control,overexpression of Mh-mi R393 inhibited the development of lesions,and inhibited endogenous Mh-mi R393a leaves from becoming more susceptible.The expression of primary auxin-responsive genes IAA8,IAA20 and GH3.7 in transiently transformed leaves was detected by RT-q PCR,the amount of H₂O₂produced was used as an indicator to measure the accumulation of ROS in transiently transformed leaves,RT-q PCR analysis of key genes in the jasmonic acid signal transduction pathway in transiently transformed leaves.The results showed that Mh-mi R393a positively regulates the resistance to apple alternaria blotch by inhibiting the expression of auxin-responsive genes,inhibiting the generation of ROS,and enhancing the defense response mediated by JA.