How transgenic plant is used in agricultural field

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How transgenic plant is used in agricultural field

  1. 1. How Transgenic plant is used in Agricultural Field 转基因植物在农业生产中的应用 Dr. Hongmei Cheng 程红梅 Biotechnology Research Institute, CAAS chenghm@caas.net.cn, Tel:82106125, Crop institue buliding 401
  2. 2. Biotechnology Research Institute was founded in 1986
  3. 3. BRI-CAAS Permanent staff: 112 Academician: 1 Professor: 26 Associate Professor: 32 Assistant Professor: 24 PhD and Master Students >150
  4. 4. Organizations-Departments and labsResearch Departments: 2Department of Plant Biotechnology and Molecular BiologyDepartment of Molecular MicrobiologyLaboratories: 8Laboratory of Plant Genetic EngineeringLaboratory of molecular biology for plant stress toleranceLaboratory of plant metabolic engineeringLaboratory of plant functional genomicsLaboratory of gene expression and molecular farmingLaboratory of biosafety assessment of GMOsLaboratory of genetic engineering for agro-microorganismsLaboratory of genetic engineering for environmental- microorganisms
  5. 5. Organizations-Research CentersResearch Centers:1. Research Center for Crop Molecular Designing2. Research Center for Microorganism Genetic Engineering3. Research Center for Biosafety Assessment of GMOs4. Key laboratory for Crop Molecular Biology, Ministry of AgricultureEngineering Center1. Center for Biotechnology Products
  6. 6. Remarkable Achievements
  7. 7. Insect Resistance Transgenic Bt cotton resistant to ball worm 1. Commercialized since 1998 2. In 2007, more than 70% cotton are transgenic, Accumulated acreage: 2.4 million ha. 3. Accumulated benefit since 1998: >126 billion RMB
  8. 8. Molecular Farming and BioreactorWithout signal peptide With signal peptide targeting to extracellular space Phytase Expression Vectors Efficiency corn transformation systemPhytase corn in greenhouse Molecular Screen and enzymatic activity assay
  9. 9. Producing phytase by transgenic corn 1. Used as feed additive to increase the efficiency use of phosphorus and proteinsmetal ions bioavailability 2. Highly expressed lines have been obtained 3. Biosafety assessment of transgenic cornPhytase corn in field completed
  10. 10. What is Biotechnology? How about some definitionsGeneral Definition The application of technology to improve a biological organismDetailed Definition The application of the technology to modify the biological function of an organism by adding genes from another organism
  11. 11. These definitions imply biotechnology is needed because:•Nature has a rich source of variation• Here we see bean has many seedcoat colors and patterns in nature But we know nature does not have all of the traits we need
  12. 12. But nature does not contain all the genetic variation man desires•Fruits with vaccines•Grains with improved nutrition
  13. 13. Central Dogma of Molecular Genetics(The guiding principle that controls trait expression) Protein Trait (or phenotype) Translation Seed shapeDNA RNA Transcription(gene) Plant height
  14. 14. In General, Plant Biotechnology Techniques Fall Into Two ClassesGene Manipulation • Identify a gene from another species which controls a trait of interest • Or modify an existing gene (create a new allele)Gene Introduction • Introduces that gene into an organism • Technique called transformation • Forms transgenic organisms
  15. 15. Genes Are Cloned Based On:Similarity to known genes Homology cloning (mouse clone used to obtain human gene)Protein sequence Complementary genetics (predicting gene sequence from protein)Chromosomal location Map-based cloning (using genetic approach)
  16. 16. Homology Cloning Clones transferred to filterHuman clone Mouse probe library added to filter Hot-spots are human homologs to mouse gene
  17. 17. Complementary Genetics1. Protein sequence is related to gene sequence NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO- ATG GAT-GCT TGG-AGT-AAA C C C G A TCT G C A G2. The genetic code information is used to design PCR primers Forward primer: 5’-ATGGAT/CGCN-3’ Reverse primer: 5’-T/CTTNC/GT/ACCA-3’ Notes: T/C = a mixture of T and C at this position; N = a mixture of all four nucleotides Reverse primer is the reverse complement of the gene sequence
  18. 18. Complementary Genetics (cont.)3. Use PCR to amplify gene fragment a. template DNA is melted (94C) 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ b. primers anneal to complementary site in melted DNA (55C) 3’ 5’ 5’ 3’ c. two copies of the template DNA made (72C) 3’ 5’ 5’ 3’
  19. 19. Complementary Genetics (cont.)4. Gene fragment used to screen library Clones transferred to filter Human clone library PCR fragment probe added to filter Hot-spots are human gene of interest
  20. 20. Map-based Cloning Gene Marker1. Use genetic techniques tofind marker near gene Gene/Marker2. Find cosegregating marker3. Discover overlapping clones (or contig) that contains the marker Gene/Marker Gene/Marker4. Find ORFs on contig5. Prove one ORF is the gene by Mutant + ORF = Wild type? transformation or mutant analysis Yes? ORF = Gene
  21. 21. Gene Manipulation• It is now routine to isolate genes• But the target gene must be carefully chosen• Target gene is chosen based on desired phenotypeFunction: Glyphosate (RoundUp) resistance EPSP synthase enzyme Increased Vitamin A content Vitamin A biosynthetic pathway enzymes
  22. 22. Introducing the Gene or Developing TransgenicsSteps1. Create transformation cassette2. Introduce and select for transformants
  23. 23. Transformation CassettesContains1. Gene of interest • The coding region and its controlling elements2. Selectable marker • Distinguishes transformed/untransformed plants3. Insertion sequences • Aids Agrobacterium insertion
  24. 24. Gene of Interest Promoter TP Coding RegionPromoter Region • Controls when, where and how much the gene is expressed ex.: CaMV35S (constitutive; on always) Glutelin 1 (only in rice endosperm during seed development)Transit Peptide • Targets protein to correct organelle ex.: RbCS (RUBISCO small subunit; choloroplast targetCoding Region • Encodes protein product ex.: EPSP -carotene genes
  25. 25. Selectable Marker Promoter Coding RegionPromoter Region • Normally constitutive ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoterCoding Region • Gene that breaks down a toxic compound; non-transgenic plants die ex.: nptII [kanamycin (bacterial antibiotic) resistance] aphIV [hygromycin (bacterial antibiotic) resistance] Bar [glufosinate (herbicide) resistance]
  26. 26. Effect of Selectable MarkerNon-transgenic = Lacks Kan or Bar Gene Plant dies in presence of selective compound X Transgenic = Has Kan or Bar Gene Plant grows in presence of selective compound
  27. 27. Insertion Sequences TL TRRequired for proper gene insertions • Used for Agrobacterium-transformation ex.: Right and Left borders of T-DNA
  28. 28. Let’s Build A Complex Cassette pB19hpc (Golden Rice Cassette) TL aphIV 35S Gt1 psy 35S rbcS crtl TRT-DNA Hygromycin Phytoene Phytoene T-DNABorder Resistance Synthase Desaturase BorderInsertion Selectable Gene of Gene of InsertionSequence Marker Interest Interest Sequence
  29. 29. Delivering the Gene to the Plant• Transformation cassettes are developed in the lab• They are then introduced into a plant• Two major delivery methods • Agrobacterium Tissue culture • Gene Gun required to generate transgenic plants
  30. 30. Plant Tissue Culture A Requirement for Transgenic Development Callus growsA plant part Shoots Is cultured develop Shoots are rooted; plant grows to maturity
  31. 31. Agrobacterium A natural DNA delivery system• A plant pathogen found in nature• Infects many plant species• Delivers DNA that encodes for plant hormones• DNA incorporates into plant chromosome• Hormone genes expressed and galls form at infection site Gall on stem Gall on leaf
  32. 32. But Nature’s Agrobacterium Has ProblemsInfected tissues cannot be regenerated (via tissue culture)into new plantsWhy? • Phytohormone balance incorrect regenerationSolution? Transferred DNA (T-DNA) modified by • Removing phytohormone genes • Retaining essential transfer sequences • Adding cloning site for gene of interest
  33. 33. The Gene Gun• DNA vector is coated onto gold or tungsten particles• Particles are accelerated at high speeds by the gun• Particles enter plant tissue• DNA enters the nucleus and incorporates into chromosome• Integration process unknown
  34. 34. Transformation StepsPrepare tissue for transformation • Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryosIntroduce DNA • Agrobacterium or gene gunCulture plant tissue • Develop shoots • Root the shootsField test the plants • Multiple sites, multiple years
  35. 35. The Lab Steps
  36. 36. Lab Testing The TransgenicsInsect Resistance Cold Tolerance Transgene= Transgene=Bt-toxin protein CBF transcription factors
  37. 37. The Next Test Is The Field Herbicide Resistance Non-transgenics Transgenics
  38. 38. Final Test Consumer Acceptance RoundUp Ready CornBefore After
  39. 39. The Organization of the CBFTranscriptional Activator ProteinEncoding Genes in Tomato
  40. 40. Plant Responses to Cold Cold-acclimating, freezing tolerant Non-acclimating, freezing intolerant Non-acclimating, chilling intolerant Presume the differences due to regulation of cold induced genes
  41. 41. Cold acclimation and freezing tolerance in Arabidopsis thaliana 2 days -5oC; 4 days recovery at 20oC No acclimation 4 days ~ 2 oC
  42. 42. Arabidopsis Genes Cold – regulated genes - COR - 1st identified low temp. induced genes
  43. 43. Function of COR15aTwo classifications –LEA II proteins –Novel hydrophilic proteins • Involves the stabilization of membranes • Decrease the propensity of membranes to from hexagonal II phase lipids in response to freezing
  44. 44. COR GenesPromoter elements – C-repeat/Drought responsive element (CRT/DRE)
  45. 45. Promoter elements of COR Genes: – C-repeat/Drought responsive element (CRT/DRE)COR genes was accomplished by overexpressing theArabidopsis transcriptional activator CBF1 (CRT/DRE bindingfactor 1)CBF1 binds to CRT/DRE DNA regulatory element present inthe promoters of the COR genesCBF1 resulted in a greater increase in freezing tolerance thandid expressing COR15a alone.
  46. 46. CBF1 pathway might control one set of cold-acclimationresponse CBF1 has a mass of 24 kDa, has AP2 domain inStockinger:Arabidopsis, tobacco, and other plants proteins. have demonstrated that Ap2 domain includes aOhme-takagiDNA-binding region.CBF1is a transcriptional activator that can activateCRT/DRE-containing genes and was a probable regulator ofCOR gene expression in ArabidposisCBF1 appears to be an important regulator of the cold-acclimation response, controlling the level of COR geneexpression.
  47. 47. CRT/DRE SequencesCOR15a ATTTCATGGCCGACCTGCTTTTT ACTTGTTGGCCGACATACATTTG CAAAATAAACCGACAAGGTTGCACOR15b ACTTGATGGCCGACCTCTTTTTT TGTGGCATACCGACTTCTAGATGCOR78 AAGATCAAGCCGACACAGACACG GATATACTACCGACATGAGTTCC AATATCATACCGACATCAGTTTG AGACATGGACCGACTACTAATAARD29b AAACGTGGACCGACTAAAACTAAKIN1 AAATAGCTACCGACATAAGGCAA ACTACTGATCCGACATCAAAACCCOR6.6 AAAAAGCTACCGACATAAGCCAACOR47 ATTCATCTACCGACTTCAAGAAA CAATCAAAGCCGACCATTCAGCT CCCACATGACCGACATCTTATGC TAGCTTTAGCCGACGTGTCTAATERD10/LTI45 ACCGACCGACCGACGTAAAAGAA ATTCATCCACCGACCGACCGACG
  48. 48. CBF Gene Family Binds CRT/DRE element Transcription activators Plays a regulatory role – Overexpression induces COR genes
  49. 49. CBF Gene FamilyCBF is a member of a small gene family encoding three closelyrelated transcriptional activator.CBF1, CBF2 and CBF3 are physically linked n direct repeaton chromosome 4 near molecular markers PG11 and m600(-71cM)Like CBF1, both CBF2 and CBF3 proteins can activateexpression of reporter genes in yeast that contain the CRT/DRE asan upstream activator sequence, indicating that these two familymember are also transcriptional activators.
  50. 50. CBF1 1 32 44 47 106 213 NLS AP2 Domain Activation Domain“Zip-Code”to get proteininto nucleus Binds to CRT/DRE “Flips the switch” Causing Gene Activation
  51. 51. COR Gene Activation by CBF Activation Domain DNA Binding Domain COR GENECRCR CR TA TA T/ T/ T/ DR DR DR E E E
  52. 52. Thomashow 2001
  53. 53. Proposed Regulatory Mechanism• Warm temperature COR
  54. 54. Proposed Regulatory Mechanism• Cold temperature CBF CBF COR
  55. 55. Proposed Regulatory Mechanism• Cold temperature CBF CBF COR
  56. 56. Project OverviewMost plants possess CBFs– Based on BLASTs of different crop speciesTomato– Cultivated species, TA491– Cold tolerance wild species, LA407
  57. 57. Chromosomal Location of the 6 Arabidopsis CBFsAt1g12610DREB1F CBF4 DREB1D At5g51990At1g63030 CBF1 CBF3 CBF2 DREB1E DREB1B DREB1A DREB1C At4g25490 At4g25480 At4g25470 1 4 5
  58. 58. CBF Signature SequencesAtCBF1: PKKPAGRKKFRETRHP FADSAWRAtCBF2: PKKPAGRKKFRETRHP FADSAWRAtCBF3: PKKPAGRKKFRETRHP FADSAWRAtCBF4: PKKRAGRKKFRETRHP FADSAWRAtCBF5: PKKRAGRRIFKETRHP FSDSAWRAtCBF6: PKKRAGRRVFKETRHP FADSAWRLeCBF1: PKKPAGRKKFRETRHP FSDSAWRLeCBF2: PKKPAGRKKFRETRHP FADSVWRGmCBF1: PKKRAGRKKFRETRHP FADSAWRGmCBF2: PKKRAGRKKFRETRHP FADSASRGmCBF3: PKKRAGRRVFKETRHP FADSRWRMtCBF3: PKKRAGRKKFKETRHP FADSAWRMtCBF2: PKKRAGRKKFKETRHP FADSAWRMtCBF1: PKKRAGRRVFKETRHP FADSAWRHvCBF1: PKRPAGRTKFHETRHP FADSAWRHvCBF3: PAKRPAGRTKFRETRHP FADSAWLConsensus: PKKPAGRKKFRETRHP FADSAWR R Rx K S
  59. 59. Objectives• Estimate CBF gene copy number• Clone all family members,• Sequence all CBFs in Tomato, and analyze it, include upstream and downstream sequence• Determine expression in response to: • Low temperature • Drought
  60. 60. Lambda Phage Clone Phage Genomic clone:Le3
  61. 61. Lambda Phage ClonePhage Genomic subclones and sequence:• Isolate le3DNA from the phage plate• Digest the DNA with NotI or Xba I enzymes• Subclone them into NotI or XbaI cut pGEM11Z• Get the physics map of Le3 19kb fragment• Sequence clones• Design primers to do the primer walk
  62. 62. Sequence analysis:• Alignment sequence data using Sequencher software• Detect the CBF loci from the sequence, find the openreading frame• Protein sequence alignment of AtCBF and LeCBF
  63. 63. 23/CHENG4/M13R.phd.121/CHENG3/M13R.phd.1 03/cheng3/E10.phd.1 43/CHENG3/ES173.phd.1 46/CHENG8/ES176.phd.1 44/CHENG3/ES204.phd.1 30/CHENG8/M13F.phd.1 31/CHENG3/ES184.phd.1 14/CHENG3/ES155.phd.1 31/CHENG3/E7.phd.1 43/CHENG3/ES203.phd.1 29/CHENG7/M13R.phd.1 30/CHENG3/ES183.phd.1 01/cheng3/E8.phd.1 28/CHENG7/M13F.phd.1 42/CHENG3/ES172.phd.1 26/CHENG6/M13F.phd.1 15/CHENG3/ES156.phd.1 22/CHENG4/M13F.phd.1 20/CHENG3/M13F.phd.1 44/CHENG6/ES174.phd.1 07_CHENG2_E23 22/CHEN2/M13F.phd.1 34/CHENG6/ES187.phd.1 02/cheng2/E9.phd.1 19/CHENG2/ES163.phd.1 06_CHENG2_E22 28/CHENG2/ES181.phd.1 27/CHENG2/E3.phd.1 05/cheng2/E12.phd.11 188 363 585 866 1,038 1,234 1,585 1,939 2,183 2,446 2,775 3,021 3,349 3,686 3,953 4,241 4,506 4,905
  64. 64. CCAAAAGGGAAGTATCAAAGTACAGAAAAAAACTAAAAATATGCCAAGTTAGACGCACGGAAGATTTGGAAGTTGAAACTTAACTTTTCTTAAACCCACAGCCCCACTCCAGCTGTCATATAAAACAGCTGCCCCACTCTATTTTTTAATAACAGCCTGTCTACTTATCACCACCCTCTAACTCCGTGTTCTTTGGTCTCAACTATATATAGAAATCAAACTTTTCACATTTTACCATAACAATTAAACTCTCTAACATCATAAATATCACTAGTTAAAGAAAGAAACAAAAATATAAATCGATATGTTTTATTCGGACCCACGTATAGAATCTTGTTCATCGTTTTCTGACAGTATTAGAGCCAATCATTCTGACGAGGAAGTTATTTTAGCTTCAAATAATCCGAAGAAGCCAGCTGGCAGAAAGAAGTTTCGAGAAACTCGACATCCAGTGTACAGGGGAGTGAGGAAGAGGAATTCTGGAAAATGGGTTTGTGAAGTCAGAGAACCAAATAAGAAGACGAGGATTTGGCTTGGTACTTTTCCTACTGCTGAAATGGCGGCTAGAGCTCATGATGTGGCGGCTATAGCATTAAGAGGACGTTCAGCTTGTTTGAATTTTGCTGACTCTGCTTGGAGGCTGCCTACTCCAGATTCCTCTGACACTAAGGATATTCAAAAGGCGGCCGCTCAGGCCGCCGAAATCTTCCGACCTTTAAAGTCGGAGGAAGAAGAATCAGTGGTTAAAGATCAATCTACTACTCCAGATGATATGTTTTTTATGGATGAGGAAGCGTTATTCTGCATGCCGGGTTTACTTACGAATATGGCGGAAGGATTAATGGTACCTCCACCTCAATGTACTGAAATGGGAGATCATGTGGAAGCTGATGATATGCCTTTATGGAGCTATTCTATATAATAAGTAAGTATAATGAGAGGAGTAACAATGCTAAGAGTGAAGTTTATTAGTTTCGTGCTTAATATTTGGATATGGTACGAATTAGTGTATAAGTATTGTAATTTGTAATGATCATGTAGATATTACTAGTATTGCTATATACTATTATAACAAAATGGTTGAAGCTAAATGAGAATCATTGGCGTATATAAGACTATTGTGTGTTTTATGACAGTTAGTCTTAGAGTTTTTTCTCATGGTTGAATTTGGTTAAGAAGCTGTTAAATGCGTTGTTCCACCAGCTTCGGAAAAACAACAGACACATACTCTAAAAAAAGCATAAAGCATTTGCTTCTGGTTTAAGCAACTGAGTGAAAAAGTAGATTTGTGGAGTATTTTTTCAAGCCGATTACTATGTCACAATCAATCAAAGAACATTGCTTATATCATAATTTTTATAAATTTTCAAAAATAAATATATCTATATATACATATAATTATTTTTTTAAAAGTTTAACATGTACACATGTTTCTCAATTTTACACGTGTGTCCGCCTATGATACAAATTTTATTAGTAATGACAATTGTAGAACTTTCTAGAATGAAATAACAATGGAGACAATTCAAATAGTTTGGAGATATATATATGTCTCAACATTGTGGTACTAATCCAATTCCAAGCATATCGATGCTGGAAATGATGCACGTGGTCCACGCGTATAATTTCCCGCGTGAGAAAATGAAAAGTAATTTATTGGAGTTGCAATAATTGATGATATAATTAACCGTCAAAAGCGTGTGTTGAGTTTTAATCAGTTATAAATTGGTACTTAGTTCACTTGTGACTTCATACATATACATATAATCATTTCAAAGGTCAATTTTCAAACTCATCTTTCAATTGGATCAAGTAGGGGGCGGCTATATATATATATATATATTGGCCTAAGAATAGAACGGCAACTTACCCTTCACCTTCCACTATCTTTTCAAAGATTCTCAATAATCAGTAGTATGATAATGAACAATGCTAAATGATCAACAAAATTTAATCAGAAATTTTAAAAAAATTGTAACGTCTCTTTTATTTTAATATAATTTTTTTTATTTTAATAAAAGAATAAAAATATAAAAAAATATCATTTTAATCAAATTCTGATCAAATTTGCTGACCATAAGAATTTTTTCATTTATTAATTAGTTTTATTCTTCATTATACTATCGTACTTATAAAAATCTCTTTCATTATGAAACTTTACATATTTACCTTTTATTTGAATAGATTATCCTAAAATTGGTCAAAAATATCTTTGTCATTATGGAACTCAAACTTCACTTTTAGGGTTGGGGTACTTATAAATATAATAGTTTGGAATAAAATTATAAATCTAATAAAATATACTTGGGTTTATATCTAGTCTCCTAAAATAGAAATAACACACACTCTCTCTCACACACACACACACTCCCCTTTTCATTCCCTTCATATTTTGTTACTCCTATTATTTTTAACTATTCTATTCTAGTCTAATTTCTCCTCTACAAAGCTTGAATCTCTAGATATAGTTATTGTCCTCAGTTATGTTATTTTCTCATTTCAAGTATTTTCAGCTACTTCCCAACATTAGAAAAGTCCATAAAATATAAATAATAATATATAAACATAAAATAAAATTAAAAATTTATTATATATAAAAAATAGTAATTTTTTTTTGGAATGAAACTTAACCAAACTCATAAAATATGCTAATTAATTAATAAGGGATATATAGGTAAATATGTATGTATGGAAGAGACATTTTAATCTTAAAAAAATAATTTTCTTCTCTGTTTCATTTTTTTAAGAAGCAGAACTTTTAGATTCTTCCCAACAACAGAACAACTGCTTCTTACTTTTTGCAAACACTTGATTTTTCAAAAAGAAAAAACATACTTTTTTCTAGGAAAAAAAAACGCTTTTGGCCTTCCAATGAATCCAATTCTAATTCAATCTTAACAAATTTAGGGTATAATCAGAAAAAAAAATATTTTTTCTTAATTTATTAAAAGTGACCAGTAAAAATGGAAATTAGATTAGAAAATATTTGTCGAATAAATAGAGACGAAGAGAGTTTAAAAAAGAAGTTGATGAATGCTGACCTTTTCCTTTGACAACTATTGGTTCAATGAATCTCCAAAGATTTATCTCTCAATTTTAAAAAATTGGTGATGACGAGATAGATGGTATAAAATAGATGCAACAAGAATAATTTTTTTTATTTTTTTTAATGTTATCATATTGAAATGACAAAGATTGGTCAGTATATATTCCAAAAAGGAAGTAAAGAGGAAAAGTTTTACAAGTCACAAGTTGCCACACGAGTTGTACGCAAATCCACTTGTCCCATAAAACAAAACAGCTGGGCTTACGCTTTTATAATCCAGCCTGTATCCTTTAATTATCACTCCGTGTTCTCTTCTCCTTTCACTATCATACTCTACTTTCCACTATAAATATATGTAACCAACACATAACACTTCTTTAACTCAACAATTATACAAATACTTTCTATTTTTAGCTCTCAACAACAATGAATATCTTTGAAACCTATTATTCAGACTCGTTAATTTTAACCGAATCATCTTCTTCTTCATCGTCATCGTCGTTTTCTGAAGAGGAAGTTATTTTAGCTTCGAATAACCCGAAAAAGCCAGCTGGCAGGAAGAAGTTTCGAGAAACACGGCATCCGATATACAGGGGAATCAGGAAGAGGAATTCAGGAAAATGGGTTTGTGAAGTCAGAGAACCAAATAAGAAGACAAGGATTTGGCTTGGTACTTTTCCTACGGCTGAAATGGCGGCTAGAGCTCATGACGTGGCGGCTTTAGCATTAAGAGGCCGTTCTGCTTGTTTGAATTTCTCTGATTCTGCTTGGAGGCTGCCTATCCCTGCTTCCTCCAACTCTAAAGATATTCAAAAGGCGGCCGCTCAGGCCGTCGAAATCTTCCGATCGGAAGAAGTTTCAGGAGAATCTCCTGAAACGTCAGAAAATGTGCAAGAGAGTAGTGACTTCGTGGATGAGGAGGCGATCTTTTTCATGCCAGGATTACTTGCAAATATGGCAGAAGGACTTATGCTACCTCCACCTCAATGTGCAGAAATGGGAGATCATTGTGTGGAAACTGATGCCTACATGATAACTTTATGGAATTATTCTATCTAAAATAGTAGTACAATTTATCAAATTACTAGGATTTAGAAGATTTTGTTAGTTTTTGGTATTCAGTATTTAGATACTAAGAATGTATATTATTAGTATTTTTATTTTGGCCAAATACATGAACATGAACAGAAACTTGTTGGGTTTTTTTACTCAGGTACCTCAACTACATCATTTTTCTATTGATTATTGAACTACACATAATTTGTTTCTTTAAAACACTGTTGGTTGATTTTGATCGACTTTTTTATTATAAATGTCTTCAATAATGTTCGAATTGTAATAATTTTGATTAAATGAATGAAGACAAACCGTGTTAATCTTAATTGTTTTCTAATGTGTTCAAATGACTTAAGTAAAACACAATTATTCTTGAACATTTTCACTATCAATTGGATTAATGAGTTGTGGAACAACATATCTATTCTCTATCAATAATCTTCACAAATCTGGTTCCACATCAGACAACAGTGTTTGTTTAAACAGAACAAATTATGGGGATTCAATGGTTCAATAGGAAAATGACGTAGTAAAGGAATCTGAAAATAAAAAAATCGAACAAATTTAGGGATCTGCTTATTGTACCGAACCATGTAGGTAGATAGTAGTGCCACCAAATAATGACACGTGTCAATGGGATGACTTGGTTTTGGCAGTAGTGAGAAGTAAAGATTAGCGTTGCAAATTTCAAGCCGTCATATTTGAATAAATGAAGTGTGGAGTGATATGACAATGTTCAATATTTTTTGCCATTCCGAGTATTGAAGAATTACAATTTCTAACTTATTTTTCGTAATTACTGAGTATCTAAATGTTAATTTTATGAATCCAATCTAAGCAAAGTTATCTGATATTGAAAAAACTTGTTTACTTAAAAACTAAGAAAACTAAAAATATATAATCCCTTCGTTCAAAAATAATGAAGGGGTGCTCAATATGAGTCAACCATATTCAAATTAAAGTTTCAATTTCAATTCCAAAGTTATTTGTCTCAACATTGAAAAATTTCAGTATTATGAAATTACAAAATAAATAAGATATAACTTTTTCATGTTTATAAACACTTTAAAACTGTAATATATAAAATATGAGTAATGAGTAGAGTGAAATATAGGAAAGTTTCCAAATATAGCTTTTAGCCTATCGTTATCTATCTAGAATGCCATTTATTGTACTACTGCCTCCTCTTTGTACGCACTATTTTGACTTGTTCTTTTCCTTCATTCGTGTACAATTTTATTTTTCCACAAAGTTTTCGTAGGTTTAGGTTAAGATAGTTAGAATTTCTTATAAATTATTTAGTTTCTTGATTCTAATTTAAGTAACACAGTTCTAATAAATACTATACGAAGTATTATAAAATAAAAAAATAAAATTTATGATTTAAAACATAATATTTGTGTGACTATAGAAGTTATGTTAGTAAATAAGTATAACATTAGTTTCTATTGGTGAATATAACAAGCAATTATTTTAGGGACAGATTAACAATGCATTCCATGTCTAAGTCCAATTCTTTTTTCCCAATAATACTTATTTCCTTTAATTTTAAAAAAAATCTCCTCTTTTTATTCTGTTTAAAAAAAATATGATTTTTTTTTTGCTAGCAACTTTTCACGTGACATGTTTAAGGCCATAATATTAAAGTGTAGTTTTATACATTTGACATAACTTTAAATTAACACTACATGATCAAAAAAATTATTTTTTAAAACTTCGTGTCAAGTTAAACTAAACCAATTTTTATAAAACGGATGAAGTATTAAATTAGATGCACACTTTATTAATCACGTGAATATAACTAGCCTAATGAGCAAGAAGACTTGTTGAAGTCAATATATATTTCATGTGGACCTTAGACAAAAATAGTTTATTACTCTTTTATATTTCAATTTACGAAATCTTAAAATTTAATATATTTGTAAACAATATACAAAAATACTTATAAGTTATAACAATTAATATTTTAAAAATATTTAAAATATAAAATTTAATAATCAAAAATATATTTATTTAAATTTTAAAATTAAAAATATATCACGTATATTGAGATCGAGAACCCAGTACAAATATTGTAGATGAGATCTATTCCTTTTAGTTAGGAAGGAAGGAAGAAGGATAGGCAAAAAGTAGAAAGTTTGCCACATCAGCAGAAAGGCTACACGAATTATACACACTTGAGACTATAAAACAGCTGTCTACTTATCACTATCCAACTCCGTGTAATACCGAACTTTTTTAAATTCAACACTTCACTTATCATATGTTTTATATATATGCATTGAGAAAATCCAATTTCATAATTCACCACAAACCCAAAAACGTCCATCCATCGTACACTACTATATTTTACTCTCTCGTCAAAATAGTATTATCATATCATGGATATCTTTGAATCCTATTATTCAAATTCTTTCGTTGAATCATTATTATCATCGTCATTATCAATATCTGATACTAATAATCTCAATCACTACTCCCCTAATGAGGAAGTTATTATTTTAGCTTCGAATAACCCGAAAAAGCCAGCTGGCAGGAAGAAGTTTCGAGAAACTCGACATCCAGTATACAGGGGAATCAGGAAGAGGAATTCAGGAAAATGGGTTTGTGAAGTCAGAGAACCAAATAAGAAGACAAGGATTTGGCTTGGTACTTTTCCTACGGCTGAAATGGCGGCTAGAGCTCATGACGTGGCGGCTATAGCATTAAGAGGCCGTTCTGCTTGTTTGAATTTCGCTGATTCAGTTTGGAGGTTGCCTATACCTGCTTCCTCCAACTCTAAAGATATTCAAAAGGCGGCCGCTGAGGCCGCCGAAATCTTTCGATCGGAAGAAGTTTCAGGAGAATCTCCTGAAACGTCAGAAAATGTGCAAGAGAGTAGTGACTTCGTGGATGAGGAAGCGCTGTTTTCCATGCCAGGATTACTTGCAAATATGGCAGAAGGACTCATGCTACCTCCTCCCCAATGTTTAGAGATCGGAGACCATTACGTTGAATTAGCTGATGTGCACGCTTATATGCCTTTATGGAATTATTCTATATAATTACAAGATTTTAATAGTCAGTATTTTAATGGTACAAATCATGTATAGGTAAATGCGTAGTAACAATATTTGAATGAAACAAAGTGAACAAGTTCATCTAATTTATCAACAGCTATATTTCATCTATCCTTAGAATTTTCAATATATTTTATATTTTGTAGATTTAGAATTAATAACGTATAACTAATATATTGTGGTTCTTAGACTGTATTGAATAAGCGGGGAGGACATTGTCAATATGAAGAGTTTGTGGAATTTATTTTCATTATTTGTTATTTTTTTTATTTTAAAGGGAAAAGACATAAAAAGAAATTTTGAATTTGGTTCGAAAACTAACTTTAGTATTTTAACTATACGGGCGTTTAAATATTTCTCTTAACATCTTCAAAATGAATTAAAAACCACCCTGAGATTACTATTCCTTTCTCACTTGTCACATCATAGCCATGTAAGTGCCACAACAACATATCAGTATCATGTCGTTAGTTATTTTCATCATTTTTCTTTAGCATTTCTTTAAAATTAATTTACACTAATTAATTAATTCAAATTGCATTTTCTAAAATTAAAATTAAAAATGTCGACACATTTTTATTTTACCCCGTACCCAACTCCCACCCACCCCCTTACTTCTTTCTTCTTCCTTCTTCTTCACCATTTCTAACTCTCAATGTTCATTTTCCCCTCTCCATATTTTTCAACTACTTCCACCATCCAAACTCCTTTCATCGTAACACTCAAATCACCGCCGAAGCTCCATTATATATATATATATATATATTAGAAGATAAACATATAGAAACATGTAGTTAAAGGAAAGAAATAAAAACAATTTACTTCTAAGAAGACGA
  65. 65.  Tomato Genomic Clone Le-3 (~19kb) LeCBF3 LeCBF1 LeCBF2 Right  Left 4.4kb 3.2kb 3.1kb 8kb NotI NotI NotI CBF1 CBF3 CBF2 2.7kb 2kb
  66. 66. Protein sequence alignment of LeCBF1.2.3LeCBF1 MNIFETYYSDSLILTESSSSSSSS--------SFSEEEVILASNNPKKPAGRKKFRETRH 52LeCBF2 MDIFESYYSNSFVESLLSSSLSISDTNNLNHYSPNEEVIILASNNPKKPAGRKKFRETRH 60LeCBF3 -----MFYSDPRIESCSSFSDSIR-------ANHSDEEVILASNNPKKPAGRKKFRETRH 48 :**:. : : * * * . .:* :********************* AP2 DomainLeCBF1 PIYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAALALRGRSACLNFSD 112LeCBF2 PVYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 120LeCBF3 PVYRGVRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 108 *:***:****************************************:***********:*LeCBF1 SAWRLPIPASSNSKDIQKAAAQAVEIFRSEEVSGESPETSENVQESSD--FVDEEAIFFM 170LeCBF2 SVWRLPIPASSNSKDIQKAAAEAAEIFRSEEVSGESPETSENVQESSD--FVDEEALFSM 178LeCBF3 SAWRLPTPDSSDTKDIQKAAAQAAEIFRPLKSEEEESVVKDQSTTPDDMFFMDEEALFCM 168 *.**** * **::********:*.****. : . *.. ..:: ..* *:****:* *LeCBF1 PGLLANMAEGLMLPPPQCAEMGDHCVETD---AYMITLWNYSI 210LeCBF2 PGLLANMAEGLMLPPPQCLEIGDHYVELADVHAYMP-LWNYSI 220LeCBF3 PGLLTNMAEGLMVPPPQCTEMGDHVEADD-----MP-LWSYSI 205 ****:*******:***** *:*** * **.***
  67. 67. Protein sequence alignment of AtCBF and LeCBFAtCBF4 MNPFYSTFPDSFLSI-SDHRSPVS--------DSSECSPKLASSCPKKRAGRKKFRETRH 51AtCBF5 MNPFYSTFPDSFLSI-SDHRSPVS--------DSSECSPKLASSCPKKRAGRKKFRETRH 51AtCBF1 MNSF-SAFSEMFG---SDYEP-----------QGGDYCPTLATSCPKKPAGRKKFRETRH 45AtCBF2 MNSF-SAFSEMFG---SDYESPVS--------SGGDYSPKLATSCPKKPAGRKKFRETRH 48AtCBF3 MNSF-SAFSEMFG---SDYESSVS--------SGGDYIPTLASSCPKKPAGRKKFRETRH 48LeCBF1 MNIFETYYSDSLILTESSSSSSSS--------SFSEEEVILASNNPKKPAGRKKFRETRH 52LeCBF2 MDIFESYYSNSFVESLLSSSLSISDTNNLNHYSPNEEVIILASNNPKKPAGRKKFRETRH 60LeCBF3 -----MFYSDPRIESCSSFSDSIR-------ANHSDEEVILASNNPKKPAGRKKFRETRH 48AtCBF6 ---------------------------------MNNDDIILAEMRPKKRAGRRVFKETRH 27 .: ** *** ***: *:****AtCBF4 PIYRGVRQRNSGKWVCEVREPNKKSRIWLGTFPTVEMAARAHDVAALALRGRSACLNFAD 111AtCBF5 PIYRGVRQRNSGKWVCEVREPNKKSRIWLGTFPTVEMAARAHDVAALALRGRSACLNFAD 111AtCBF1 PIYRGVRQRNSGKWVSEVREPNKKTRIWLGTFQTAEMAARAHDVAALALRGRSACLNFAD 105AtCBF2 PIYRGVRQRNSGKWVCELREPNKKTRIWLGTFQTAEMAARAHDVAAIALRGRSACLNFAD 108AtCBF3 PIYRGVRRRNSGKWVCEVREPNKKTRIWLGTFQTAEMAARAHDVAALALRGRSACLNFAD 108LeCBF1 PIYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAALALRGRSACLNFSD 112LeCBF2 PVYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 120LeCBF3 PVYRGVRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 108AtCBF6 PVYRGIRRRNGDKWVCEVREPTHQRRIWLGTYPTADMAARAHDVAVLALRGRSACLNFAD 87 *:***:*:**..***.*:***.:: ******: *.:*********.:***********:*
  68. 68. AtCBF4 SAWRLRIPETTCPKEIQKAASEAAMAFQNETTT---EGS-KTAAEAEEAAGEGVREGERR 167AtCBF5 SAWRLRIPETTCPKEIQKAASEAAMAFQNETTT---EGS-KTAAEAEEAAGEGVREGERR 167AtCBF1 SAWRLRIPESTCAKDIQKAAAEAALAFQDETCD---TTTTDHGLDMEETMVEAIYTPE-- 160AtCBF2 SAWRLRIPESTCAKEIQKAAAEAALNFQDEMCH---MTTDAHGLDMEETLVEAIYTPE-- 163AtCBF3 SAWRLRIPESTCAKDIQKAAAEAALAFQDEMCD---ATT-DHGFDMEETLVEAIYTAE-- 162LeCBF1 SAWRLPIPASSNSKDIQKAAAQAVEIFRSEEVS---GESPETSENVQE------------ 157LeCBF2 SVWRLPIPASSNSKDIQKAAAEAAEIFRSEEVS---GESPETSENVQE------------ 165LeCBF3 SAWRLPTPDSSDTKDIQKAAAQAAEIFRPLKSE---EEESVVKDQSTT------------ 153AtCBF6 SAWRLPVPESNDPDVIRRVAAEAAEMFRPVDLESGITVLPCAGDDVDLGFGSGSGSGSGS 147 *.*** * :. .. *::.*::*. *: :AtCBF4 AEEQNGGVFYMDDEALLGMPNFFENMAEGMLLPPPE---VGWNHN-DFDGVGD----VSL 219AtCBF5 AEEQNGGVFYMDDEALLGMPNFFENMAEGMLLPPPE---VGWNHN-DFDGVGD----VSL 219AtCBF1 ---QSEGAFYMDEETMFGMPTLLDNMAEGMLLPPPS---VQWNHNYDGEGDGD----VSL 210AtCBF2 ---QSQDAFYMDEEAMLGMSSLLDNMAEGMLLPSPS---VQWNYNFDVEGDDD----VSL 213AtCBF3 ---QSENAFYMHDEAMFEMPSLLANMAEGMLLPLPS---VQWNHNHEVDGDDDD---VSL 213LeCBF1 ----SSD--FVDEEAIFFMPGLLANMAEGLMLPPPQ---CAEMGDHCVETD---AYMITL 205LeCBF2 ----SSD--FVDEEALFSMPGLLANMAEGLMLPPPQ---CLEIGDHYVELADVHAYMP-L 215LeCBF3 ----PDDMFFMDEEALFCMPGLLTNMAEGLMVPPPQ---CTEMGDHVEADD-----MP-L 200AtCBF6 EERNSSSYGFGDYEEVS---TTMMRLAEGPLMSPPRSYMEDMTPTNVYTEEEMCYEDMSL 204 . : . * : : .:*** ::. * *
  69. 69. Drought Nacl(250mm) ABA(100µm) McM 1 2 4 6 8 24 1 2 4 8 16 24 1 2 4 8 16 24 (hours)3’ LeCBF1.3’ LeCBF23’ LeCBF3 Le25 eIF4A cold 4ºC (24hL) cold 4ºC(16hL/D) Control 0 .25 .5 1 2 4 8 24 7d 0 1 2 4 8 16 24 0 1 2 4 8 16 24 (hours)3’ LeCBF13’ LeCBF23’ LeCBF3 eIF4A
  70. 70. Tomato CBF Genes Expression Pattern Under Different Stress The expression of LeCBF1 and LeCBF2 under various stress treatments was investigated using RNA Gel Blot analysis LeCBF1 but not LeCBF2 was found to be cold-responsive Drought Nacl(250m ) m ABA(100µm) McM 1 2 4 6 8 24 1 2 4 8 16 24 1 2 4 8 16 24 (hours) 3’ LeCBF1. 3’ LeCBF2 Le25 eLF4A cold 4ºC(24hL) cold 4ºC(16hL/D) Control 0 .25 .5 1 2 4 824 7d 0 1 2 4 8 1624 0 1 2 4 8 16 24 (hours) 3’ LeCBF1 3’ LeCBF2 eLF4A
  71. 71. Thank You!

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