| ETI is an important mechanism which plant resist to pathogen in the long term evolutionary process.The interaction between tomatoes and Cladosporium fulvum is one of the model systems to study gene for gene hypothesis,which results from ETI.Several Cf genes and their complementary avirulence genes(Avr)have been cloned.In addition,study on their interaction mechanisms and signaling pathways in Cf-2,Cf-4 and Cf-9 has been focused on and made some progress.Although these R gene sequences are highly identical,they have distinct interaction mechanisms.Cf-4E and Avr4 E are a pair of complementary genes,however,the mechanism of interaction and signaling pathway determined by Cf-4E/Avr4 E are less known.Therefore,the study mechanism of this complementary gene pair can enrich the theoretical understanding of the signal pathway of Cf/Avr interaction,but also provide a theoretical basis for disease resistance breeding.A single copy transgenic Nicotiana.benthamiana line,which carried both Avr4E-driven by 35 s and Cf-4E-driven by promoter-induced with estradiol,has been acquired in the previous a few study.No one of seeds from this material geminates due to strong HR in the presence of estradiol.However,if any one of Cf-4E,Avr4 E or key signal component was mutated,the seeds will germinate and grow on medium with estradiol.On the contrary,if none of these genes is mutated,seeds will not germinate on medium with estradiol.In view of the above-mentioned,research about mutant induced by EMS was carried out in this study,(1)If the chemical mutagen was used to generate mutation,different concentration had the distinct effect on result and optimal concentration is also different in varied plants seeds.This mean optimal result strictly depend on both concentration of mutagenic agent and species.So the evaluation of semi-lethal concentration of EMS for N.benthamiana was performed.According to statistic result,the lethal concentration was 0.6% and LC50 of EMS was 0.34%.As a selection criterion,the suitable concentration of EMS mutagenized seeds was determined to be 0.35%.(2)In order to increase the mutation rate,the transgenic seeds were treated with 0.35% EMS mutagenesis,223 seeds geminated in total 320,000 seeds treated by EMS under the estradiol and these seedlings were all transferred to pot with nutrient soil.Most of the seedlings grow strictly and died before flowering.The remaining 87 seedlings can normally grow,most of which displayed phenotypes such as albinism,clump,yellowing,slender,dwarf,leaf blade deformity and so on.Injecting estradiol on their leaves,62 plants appeared HR on injecting area and 27 plants,which did not display HR on injecting area,were confirmed candidate targets.However,only 12 plants of these candidates were able to produce seeds.In 12 lines,5 lines,whose seeds can geminate on medium with estradiol,kanamycin and hygromycin,were thought as positive targets.They were TB-9.2,TB-12.14,TB-19.1,TB-22.1,TB-31.2.To confirm the authenticity of the mutants and the possible types of mutations,agrobacterium-mediated transient expression was employed.If HR did not appeared upon injecting both Cf-4E and Avr4 E,the mutation occurs in components in signal transduction pathway rather than the Cf-4E and Avr4 E genes.On the contrary,mutation happened on Cf-4E and/or Avr4 E.If HR appeared with injection of estradiol,indicating a false positive plant.According to result agrobacterium-mediated transient and injecting estradiol,we speculated Cf-4E was possibly mutated in TB-9.2 and Avr4 E was done in TB-22.1.In TB-12.14 and TB-19.1,possible key signal components were mutated.Cf-4E from TB-9.2 was amplified by pcr and sequenced,suggesting Cf-4E is not mutated.We thought mutation occur in inducible promote region and lead to lost ability of response to estradiol. |
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