Identification Of Isolates Of Hyaloperonospora Parasitica And Cloning And Expression Analysis Of BcOPR3 Gene In Non-heading Chinese Cabbage

Non-heading Chinese cabbage(Brassica rapa ssp.Chinensis),which origin from China,is one of the most extensively cultivated vegetables in China,and play a vital role in the vegetable market in China.Downy mildew is the second largest disease in non-heading Chinese cabbage,causing severe reductions in crop yield and declining the quality of non-heading Chinese cabbage.Therefore,disease resistance is an important breeding target of non-heading Chinese cabbage,and developing resistant cultivars is an effective prevention.At present,although a number of resistant cultivars have been selected,the mechanism of resistance is still unknown,which greatly limits the process of molecular breeding of non-heading Chinese cabbage resistance to disease.In this paper,identification of isolates of Hyaloperonospora parasitica from non-heading Chinese cabbage and cloning and expression analysis of BcOPR3 gene in non-heading Chinese cabbage was studied.In the study,the main conclusions were as follows:1.Identification of isolates of Hyaloperonospora parasitica from non-heading Chinese cabbageIn this study,to identify the 10 isolates from non-heading Chinese cabbage downy mildew lesions,random amplification of polymorphic DNA(RAPD)was evaluated as an effective method for typing strains of non-heading Chinese cabbage downy mildew.The similar coefficient between 10 isolates ranged from 0.125 to 0.75,with the phenon line(0.56)delimiting three groups.To analyze the specific immunity-related expression of gene during downy mildew infection,RT-PCR experiments focusing on 18 genes related to immunity were conducted.The results showed that the genes with similar function could not be clustered together after the infection of the downy mildew pathogen,and 8 genes(45%)of the 18 genes were down-regulated at the transcriptional level.One gene(6%)was up-regulated;4 genes(22%)were rapidly up-regulated at the transcriptional level,and then slowly down-regulated,finally,kept stable;2 genes(11%)were rapidly down-regulated,then rapidly up-regulated and down-regulated,finally,rapidly up-regulated;3 genes(17%)were rapidly down-regulated and then rapidly up-regulated at the transcriptional level.Hierarchical clustering of these expression patterns provide information about which genes are co-regulated over the time of plant immune response.2.Expression analysis of BcOPR3 gene in non-heading Chinese cabbageThe 12-oxo-phytodienoic acid reductase(OPR)is a key enzyme in the biosynthesis pathway of jasmonic acid,which catalyzes the reduction of OPDA to produce jasmonic acid compounds,and it is widely involved in the process of plant growth anddefense system.Clone and identify the OPR3 gene of non-heading Chinese cabbage,and the signal molecular and abiotic stress response was important to the study of non-heading Chinese cabbage resistance mechanism.To explore the function of the OPR3 gene in non-heading Chinese cabbage,we cloned BcOPR3 and conducted bioinformatics analysis.Its sub-cellular localization was researched.We also analyzed the expression level of BcOPR3 under Hyaloperonospora parasitica,ABA,JA,SA,NaCl,mechanical injury,hypothermia and heat shock stress treatments.The sequence of BcOPR3 was consistent with four kinds of non-heading Chinese cabbage.The homology between Brassica campestris ssp.chinensis Makino and Brassica napus was up to 98%,according to the close relationship.BcOPR3 was verified to be located in the cytoplasm.Quantitative real-time RT-PCR analysis showed that Hyaloperonospora parasitica,ABA,JA,SA,NaCl,mechanical injury,hypothermia and heat shock stress induced the expression of BcOPR3.The BcOPR3 gene was cloned from non-heading Chinese cabbage.The BcOPR3 gene may co-respond to Hyaloperonospora parasitica,ABA,JA,SA,NaCl,mechanical injury,hypothermia and heat shock stress.In non-heading Chinese cabbage,the gene plays an important role in the cytoplasm.The BcOPR3 gene had a different expression between resistant line'Suzhouqing' and susceptible inbred line 'Aijiaohuang' and it had a higher expression on the 'Suzhouqing' than the 'Aijiaohuang' under downy mildew infection.The high transcription level of BcOPR3 is an important reason that resistant line has a higher resistance than the susceptible under downy mildew infection.BcOPR3 has a different expression pattern under abiotic stress,and it may be involved in different signaling pathways.