Functional Analysis Of Malus Hupehensis MiR397b In Response To Botryosphaeria Dothidea

Apple(Malus×domestica Borkh)is one of the most important fruit tree species in the world.Due to its wide cultivation area,it is inevitably subjected to various biotic and abiotic stresses,especially pathogen-induced plant diseases.Malus hupehensis(Pamp.)Rehd is a wild germplasm of the genus Rosaceae in China.It is widely used as a stock for apples because of its strong resistance to stress.Apple ring rot is a fungal disease caused by Botryosphaeria dothidea and is one of the most serious diseases in apple production in China.MiRNA-mediated post-transcriptional regulation plays a crucial role in plants response to pathogens.MiR397 is a highly conserved miRNA in flowering plants and one of the most common miRNAs regulating plant life processes,although miR397 plays an important regulatory role in plant growth and development and abiotic stress response,little is known about the functional studies of miR397 in plant resistance to pathogens.In this study,we analyzed the expression levels of mature Mh-miR397 and MhLAC7,lignin content and laccase activity in M.hupehensis infected with B.dothidea,and evaluated the resistance of transiently expressed Nicotiana benthamiana leaves to Botrytis cinerea and M.hupehensis to B.dothidea to determine the mechanism of Mh-miR397b in plant response to pathogens resistance.The main results are as follows:1.In order to explore the expression regulation pattern of Mh-miR397 and its predicted target genes in response to B.dothidea infection,the leaves of M.hupehensis were inoculated with B.dothidea and subsequently colleted in different time points,the expression levels of mature Mh-miR397 and its predicted target genes were analyzed by RT-qPCR,the lignin content and laccase activity were also measured.The results showed that the expression of Mh-miR397 and its predicted target MhLAC7 could be induced and both of them showed a certain negative correlation in M.hupehensis response to B.dothidea.In addition,the dynamic trends of lignin content and laccase activity showed a certain positive correlation with the trend of MhLAC7 expression.2.In this study,the precursor sequence of Mh-miR397b was cloned from the genomic DNA of M.hupehensis,and the MhLAC7 gene was cloned from the cDNA obtained from the reverse transcription of total RNA from the leaves of M.hupehensis.The interaction between Mh-miR397 and MhLAC7 was analyzed by 5’RLM-RACE and tobacco co-transformation.5’RLM-RACE results indicated that MhLAC7 was cleaved by Mh-miR397 in plants and the cleavage site occurred between the 10th and 11th bases of the Mh-miR397 target site.The histochemical staining results and GUS activity assay in tobacco co-transformation indicated that Mh-miR397b could inhibit the expression of GUS gene in MhLAC7.Thus,these results demonstrated that Mh-miR397b can regulate the expression of MhLAC7 by base pairing.3.In this study,the transient expression system of N.benthamiana and M.hupehensis was constructed to analyze the resistance of transiently expressed N.benthamiana leaves to Botrytis cinerea and transiently expressed M.hupehensis leaves to B.dothidea.The results showed that Mh-miR397b negatively regulates the resistance of N.benthamiana to B.cinerea by regulating the exogenous MhLAC7 and endogenous NtLAC7 involved in lignin biosynthesis in N.benthamiana,Mh-miR397b negatively regulates the resistance of M.hupehensis to B.dothidea by regulating the expression of MhLAC7 involved in lignin biosynthesis.