| Malus domestica Borkh belongs to Malus genus in the family of Rosaceae,with variations of ecological adaptability,nutritional value,and long shelf life etc.In the world,many countries list apple as the main fruit crop in the world.China is the major apple production center in the world which accounted for more than 40%of the total output.Apple leaf spot is a worldwide disease which is prevalent in the world apple production area.The disease heavily reduces the quality and yield of apple,and cause serious early defoliation.Therefore,it is of great significance to explore breeding for resistant cultivars.Apple blotch,caused by Alternaria alternata apple pathotype,is one of the worldwide diseases which occur in major apple production areas,particularly in East Asia countries including China.Thus enhancing the resistance to apple blotch is one of the important objectives of the current research.ASR genes have been extensively studied in recent years.Since the first gene was cloned in tomato,ASR genes in other species have also beenclone.However these studies mainly focused on their roles in abiotic stress,and less on biotic stresses.In this research,we studied their roles in biotic stress and attempted to find some highly expressed ASR genes in response to apple blotch infection.Research includes the following three major aspects:Firstly,identification of ASR genes in whole apple genome,using bioinformatics methods,drawing the phylogenetic tree,analyzing its physicochemical properties,structure,motif,gene structure and predicting its subcellular localization and protein function.Secondly,through artificial inoculation of'Golden Delicious' and 'Starking' leaves,we observed the morphological changes of Alternaria.alternata by microscope and recorded expansion of leaf lesion.We also determined activities of five defensive enzymes activity,POD,SOD,PAL,PPO and CAT were measured.We further used fluorescence quantitative RT-PCR to analyze expressed patterns of apple ASR gene family members in response to pathogen A.alternata infection in 'Golden Delicious' and 'Starking'.The main results are as follows:1.Five ASR genes were found in apple genome.According to their positions on the chromosome,they were named ASR1,2,5,4 and ASR5.ASR proteins in apple are highly hydrophilic protein,rich in glutamic acid,histidine,glycine,lysine and alanine.Alpha helix is the main secondary structure.Three genes are predicted to be located in nucleus.From the Ka/Ks,the value of ASR2/ASR3 gene pair(2.07)is greater than the value for the other three gene pairs(0.67,0.47 and 0.71),indicating that ARS2 and ASR3 were under positive selection.From the point of view of Ks,the value of ASR2/ASR3(0.05)is less than the other two values(0.74 and 1.51)suggesting these genes are likely to be a recent replication.From phylogenetic tree,apple family members gathered together and showed interspecific replication specificity.2.Based on the incidence and disease index,'Golden Delicious ' is considered to be have some levels of resistance to pathogen expansion whereas 'Starking' is considered to be have some levels of resistance to pathogen infection under artificial infection.The overall activitivities of five defensive enzymes tended to increase first and then decrease.The expression patterns of ASR genes varied.In 'Golden Delicious',ASR1 was up-regulated at all time points and the other four genes were down-regulated at the time point of 18h and 36h,then up-regulated at 72h.In 'Starking ',ASR1,2 and 3 were down-regulated at all time points.ASR4 and ASR5 were up-regulated at 36h and down-regulated at 18h and 72h,and their expression levels were quite low.The change of these genes showed that ASR genes may be somehow involved in the response to apple blotch infection... |
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