Screening Of Mutants Resistant To TOR Inhibitors And Preliminary Mapping In Arabidopsis Thaliana

TOR(target of rapamycin)kinase is a conservative regulatory factor in yeast,plants,animals and human evolution,it integrated the nutrition and energy signal to promote cell proliferation and growth,the TOR signaling pathway is closely related to many and biological physiological functions,such as cell growth,cell division,cell metabolism.TOR was first identified in budding yeast genetic mutation screening of rapamycin,which is a kind of immune inhibitor can block the human T cell activation and proliferation,rapamycin can combine with FKBP12 and formed RAPA-FKBP12 binary compound,then RAPA-FKBP12 complex combined with FRB domain to inhibit TOR kinase activity.Although two TOR genes identified in yeast,but then in Arabidopsis thaliana,green algae,most animals and humans only identified one TOR gene.Arabidopsis thaliana and human TOR contain highly similar amino acid sequence,especially in protein kinase region(of 75% similarity),showed that the protein kinase had similar function and protein substrate.About TOR signaling pathways in yeast in animals,people had carried on thorough researches,but as a result of Arabidopsis thaliana FKBP12 cannot form binary polymer with rapamycin,leading to rapamycin can't combine with Arabidopsis TOR FRB domain structure,and so cannot inhibit the TOR protein activity,so the research on the TOR pathway in Arabidopsis thaliana is less,this article through methyl sulfonic acid ethyl ester(EMS)induction of Columbia-0(Col-0,Col,WT)Arabidopsis chosed TOR inhibitors of resistant mutants with the mutant phenotype observation analysis and gene mapping and related research.Main conclusions are as follows:1.Mutant screening resultsColumbia(Col)Arabidopsis thaliana used in experiment had the yeast protein FKBP12 transferred into,the plant transferred into was named BP12-2.The first generation of TOR inhibitors was rapamycin(RAP),the second generation inhibitors including KU,AZD,Torin1,with RAP and AZD respectively to combine with TOR FRB domain and Kinase domain.Using EMS mutagenesis Col type of Arabidopsis thaliana,with RAP + AZD double inhibit to restrain activity of TOR,screened out 9 mutants which is not sensitive to RAP + AZD inhibitors,named TOR inhibitors resistant mutants(TOR-inhibitor-insensitive,trin),respectively,trin-1,trin-2,trin-6,trin-8,trin-10,trin-12,trin-16,trin-17,trin-19,through map-based cloning we positioned 9 mutants respectively.2.The phenotypic observationsAfter physiological maturity,9 mutants showed different phenotypes,and roughly divided into three categories:(1)no phenotypic plant: trin-1?trin-6?trin-8?trin-10 ?trin-12;(2)double fruit pods and grow slowly plant: trin-2;(3)double fruit pods sterile plant: trin-16?trin-17?trin-19.3.Potassium experimental resultsIdentified with different concentrations of potassium medium,on the basis of potassium concentration in the MS culture medium formula seven levels were set up,from low to high respectively is 0%,10%,20%,30%,20%,50% and 100%,each mutant under different concentrations of potassium were set the repeat experiments twice,with 9 mutants do direct training on different concentrations of potassium medium and the normal 1/2 MS culture medium about 10 days first,then turned into different concentration gradient of potassium medium indirect cultivation experiment,trin-2 mutant was found in 9 mutants sensitive to low potassium or without potassium.4.Genetic analysisStatistics and observation of the phenotype with mutant and wild type Lansberg(Ler)hybrid combinations in the F1 and F2 plants in culture medium,haven't shown mutant traits,found all the F1 showed wilting.wilting showed that 9 mutants were all recessive mutants;And the F2 generation showed two phenotypes with prospect in in normal growth and withering yellow filter on the medium,statistical cross combinations of different phenotypes,?2 test showed that the separation ratio of the mutant plants and normal plants is 1:3,agree that each mutant mutation trait was controlled by 1 recessive nuclear gene.5.Preliminary location resultsUsing the F2 hybrid combinations population of mutants and wild type Lansberg(Ler)to locate TOR inhibitors series of resistant mutants.The results show that trin-1 mutant position near ciw11 molecular marker at 9.3 M in the third chromosome.trin-2 mutant position between PHYC molecular marker at 13.4 M and ciw9 molecular marker at 16.3M in the fifth chromosome.trin-6 mutant position between nga280 molecular marker at 20.0M and nga111 molecular marker at 26.1M in the first chromosome.trin-8 mutant position between ciw4 molecular marker at 18.0 M and nga6 molecular marker at 22.0M in the third chromosome.trin-10 mutant position between ciw5 molecular marker at 0.7M and ciw6 molecular marker at 7.5M in the fourth chromosome.trin-12 mutant position between ciw5 molecular marker at 0.7M and ciw6 molecular marker at 7.5M in the fourth chromosome.trin-16 mutant position between PHYC molecular marker at 13.4 M and ciw9 molecular marker at 16.3M in the fifth chromosome.trin-17 mutant position between PHYC molecuLar marker at 13.4 M and ciw9 molecular marker at 16.3M in the fifth chromosome.trin-19 mutant position between PHYC molecular marker at 13.4 M and ciw9 molecular marker at 16.3M in the fifth chromosome.6.Candidate gene analysisSequenced the gene between the preliminary positioned molecular markers,we focused the genes range of mutations.Locking trin-1 mutant within eight genes;Locking trin-2 mutant within eight genes within five genes;Locking trin-6 mutant within six genes;Locking trin-8 mutant within six genes;Locking trin-10 mutant in two genes;Locking trin-12 mutant within 10 genes;Locking trin-16 mutant within 10 genes;Locking trin-17 mutant within five genes;Locking trin-19 mutant within nine genes.