| Pathogen and insect can cause 30-40% decrease of global crop production. The employment of broad-spectrum resistance varieties can not only prevent diseases and insects but also greatly reduce the cost of agricultural production and the environmental pollution caused by pesticide spray. In this experiment, Arabidopsis RNAi mutant library was screened by using Phytophthora infestans T124 as inoculums to obtain interested mutant. Through double staining and microscopic examination methods, two positive Arabidopsis plants were obtained, which show resistance to P. infestans different from the susceptibility of wild type. The silenced genes were cloned and sequenced from genomic DNA of two mutants by PCR method, BLAST analysis showed that two mutants silenced the same gene, ent-kaurene oxidase gene (KO). The mutant was named as cko. To further verify the tolerance of cko to biotic and abiotic stresses of the gene, several pathogens were used to infect cko, and salt and drought stresses were tested as well. The main results were described as follows:(1) Restore wild-type phenotype through adding GA in cko-the mutant with adding GA showed susceptible to T124Using an in-bottle detection system, WT and cko germinated on MS medium with GA for 2 weeks, then inoculation with T124 and 9 days after inoculation the pathogen T124 was observed on the mutant. The results showed that GA could restore the mutant to the wild-type phenotype.(2) cko responses a defense against the necrotrophic pathogens-Verticillium dahliae, Fusarium solani and Botrytis cinerea.Take a bottle and a detached leaves detection system, WT and cko inoculation with Verticillium dahliae, Fusarium solani and Botrytis cinerea respectively by artificial. The cko shown an obvious resistance to the three necrotrophic pathogens while WT susceptible to all the three after 7d,5d and 5d respectively.(3) cko shows a susceptible to the living parasitic fungi-Oidium neolycopersici the same as WTTake a bottle detection system,5d later, WT and cko which inoculation with Oidium neolycopersici by artificial were both observed spore germination under microscope after double stain.15d later, macroscopic Oidium neolycopersici we could see in both WT and cko.(4) cko enhanced the tolerance to salt stressesTake the WT, ckol and cko2 seeds on the same MS medium with 1% Nacl. Then germinated in a light growth chamber after 3d vernalization. Seeds from ckol and cko2 showed higher germination rates about 99% while WT lower after 9d.(5) cko enhanced the tolerance to drought stressesThe WT and cko seedlings are cultivated in soil, begin to stop watering and adding nutrient solution of several stage after 3 weeks. Under drought stresses, cko showed better performance than WT, with higher chlorophyll content.In general, through screening the Arabidopsis RNAi mutant library, two mutants with silencing the same gene were obtained, which was named as cko and showed different phenotype to Phytophthora infestans from wild type, the silenced gene was proved to be ent-kaurene oxidase gene (KO). KO is a key gene of synthesis of gibberellins (GA), adding the GA could restore the mutant to wild type phenotype to P. infestans. The mutant of cko showed resistance to several necrotrophic pathogens while wild type plants were susceptible to the pathogens. The mutant showed the same phenotype to biotrophic pathogen as wild type plants. The mutant enhanced the tolerance to salt and drought stresses compared to wild type. It demonstrated that KO gene was the key gene in the responses to necrotrophic pathogens and abiotic stresses of salt and drought, further implying that G A regulated these responses of Arabidopsis. |
PDF Full Text Request