| Since tobacco has high economic worth,large amount of germplasm resource,easy hybridization between different varieties and lots of maker genes carried by chromosomes,it is widely used as a model plant in plant pathology.Nicotiana tabacum L.belongs to a natural allotetraploid.It has a huge number of genome and high ratio of repeat sequences.Size of its genome is about 4.5 Gb containing T and S genomes.Till now,the draft genomes of N.tabacum and its ancestor,N.sylvestris and N.tomentosiformis have been assembled.This work provided the foundation to apply reverse genetic to functional genome.Building and screening the mutant library is the most efficient and direct way to search a functional gene.In this study,the T-DNA insertion mutant library was provided by China Agriculture Academy of biological technology.Here,we screened mutants with two compounds instead of pathogens because it was much easily performed.As a result,we got a set of mutants with different sensitivity to these two compounds.Moreover,one mutant of them was systematically studied.The main results are as follows:Sixteen mutants were found to have different sensitivity of salicylic acid.Salicylic acid is an important transductive signal in defending the disease.Changes in tolerance are always related to the mutation in gene which is essential to the resistance signaling pathway mediated by salicylic acid.To isolate different sensitivity mutants from 7776 varieties,we have conducted a series of work including primary screening,re-screening and confirming.Finally,we got 10 mutants more tolerant to salicylic acid and 6 mutants were more sensitive to it.Nine mutants were found to be more tolerant to oxidative stress.AT(Amitrole)is one irreversible and specific catalase inhibitor.As a product,it is transfered within the suction type herbicide absorbed by root and leaf.This makes it a simple and powerful approach for isolation of mutants with increased tolerance to oxidative stress.Using this approach we tried to find a functional gene.After screening of 7776 mutants,we got,3 mutants with increased tolerance to AT and,6 mutants were more sensitive to it.A total of 25 mutants above were identified as T-DNA insertion mutants.When inoculated with pathogens,some had big changes in ability of resistance providing the precious materials for searching functional genes.One mutant was more sensitive to AT with improved disease resistance.We selected 21AS for further studies as it was more sensitive to AT.In morphology,leaves of 21AS were ovate?the surface of leaves was uneven and leave apex was obtuse.When inoculated with Botrytis cinerea and Phytophthora parasitica,it showed obvious resistance to both pathogens.Two defense genes(NtGST1 and NtCAT1)were selected and transcriptionally analyzed.NtGST1 was up-regulated in tendency while NtCATl was down-regulated during infection with higher expression level in 21AS than WT.The level of accumulated active oxygen declined significantly in the early when induced by flg22.Interestingly,the level of cell death was also decreased when treated with cell death elicitors.It could be concluded that 21AS has an apparent change in resistance to disease,expression of defense genes and tolerance to oxidative stress.However,further study is necessary for the concrete mechanism and links.Expression characteristics and functions of PsAAT3 in Phytophthora sojae were studied.Here we conducted some experiment on one gene,PsAAT3 involved in metabolism of amino acids in Phytophthora sojae.It is an aspartate aminotransferase involved in biosynthesis of the aspartate-derived essential amino acids.PsAAT3 plays an important role in nitrogen metabolism.The results showed expression of the gene cannot be induced by starvation.Contents of free amino acid in PsAAT3-silenced mutants had no difference with wild type.But Phytophthora sojae pathogenicity was weakened after silencing of PsAAT3.And the deficiency in pathogenicity of PsAAT3-silenced mutants cannot be remedied by adding exogenous aspartate and glutamate. |
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