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How transgenic plant is used in agricultural field
- 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. Biotechnology Research Institute was founded in 1986
- 3. BRI-CAAS Permanent staff: 112 Academician: 1 Professor: 26 Associate Professor: 32 Assistant Professor: 24 PhD and Master Students >150
- 4. Organizations-Departments and labs Research Departments: 2 Department of Plant Biotechnology and Molecular Biology Department of Molecular Microbiology Laboratories: 8 Laboratory of Plant Genetic Engineering Laboratory of molecular biology for plant stress tolerance Laboratory of plant metabolic engineering Laboratory of plant functional genomics Laboratory of gene expression and molecular farming Laboratory of biosafety assessment of GMOs Laboratory of genetic engineering for agro-microorganisms Laboratory of genetic engineering for environmental- microorganisms
- 5. Organizations-Research Centers Research Centers: 1. Research Center for Crop Molecular Designing 2. Research Center for Microorganism Genetic Engineering 3. Research Center for Biosafety Assessment of GMOs 4. Key laboratory for Crop Molecular Biology, Ministry of Agriculture Engineering Center 1. Center for Biotechnology Products
- 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. Molecular Farming and Bioreactor Without signal peptide With signal peptide targeting to extracellular space Phytase Expression Vectors Efficiency corn transformation system Phytase corn in greenhouse Molecular Screen and enzymatic activity assay
- 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 corn Phytase corn in field completed
- 10. What is Biotechnology? How about some definitions General Definition The application of technology to improve a biological organism Detailed Definition The application of the technology to modify the biological function of an organism by adding genes from another organism
- 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. But nature does not contain all the genetic variation man desires •Fruits with vaccines •Grains with improved nutrition
- 13. Central Dogma of Molecular Genetics (The guiding principle that controls trait expression) Protein Trait (or phenotype) Translation Seed shape DNA RNA Transcription (gene) Plant height
- 14. In General, Plant Biotechnology Techniques Fall Into Two Classes Gene 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. 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. Homology Cloning Clones transferred to filter Human clone Mouse probe library added to filter Hot-spots are human homologs to mouse gene
- 17. Complementary Genetics 1. 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 G 2. 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. 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. 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. Map-based Cloning Gene Marker 1. Use genetic techniques to find marker near gene Gene/Marker 2. Find cosegregating marker 3. Discover overlapping clones (or contig) that contains the marker Gene/Marker Gene/Marker 4. Find ORFs on contig 5. Prove one ORF is the gene by Mutant + ORF = Wild type? transformation or mutant analysis Yes? ORF = Gene
- 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 phenotype Function: Glyphosate (RoundUp) resistance EPSP synthase enzyme Increased Vitamin A content Vitamin A biosynthetic pathway enzymes
- 22. Introducing the Gene or Developing Transgenics Steps 1. Create transformation cassette 2. Introduce and select for transformants
- 23. Transformation Cassettes Contains 1. Gene of interest • The coding region and its controlling elements 2. Selectable marker • Distinguishes transformed/untransformed plants 3. Insertion sequences • Aids Agrobacterium insertion
- 24. Gene of Interest Promoter TP Coding Region Promoter 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 target Coding Region • Encodes protein product ex.: EPSP -carotene genes
- 25. Selectable Marker Promoter Coding Region Promoter Region • Normally constitutive ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter Coding 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. Effect of Selectable Marker Non-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. Insertion Sequences TL TR Required for proper gene insertions • Used for Agrobacterium-transformation ex.: Right and Left borders of T-DNA
- 28. Let’s Build A Complex Cassette pB19hpc (Golden Rice Cassette) TL aphIV 35S Gt1 psy 35S rbcS crtl TR T-DNA Hygromycin Phytoene Phytoene T-DNA Border Resistance Synthase Desaturase Border Insertion Selectable Gene of Gene of Insertion Sequence Marker Interest Interest Sequence
- 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. Plant Tissue Culture A Requirement for Transgenic Development Callus grows A plant part Shoots Is cultured develop Shoots are rooted; plant grows to maturity
- 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. But Nature’s Agrobacterium Has Problems Infected tissues cannot be regenerated (via tissue culture) into new plants Why? • Phytohormone balance incorrect regeneration Solution? Transferred DNA (T-DNA) modified by • Removing phytohormone genes • Retaining essential transfer sequences • Adding cloning site for gene of interest
- 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. Transformation Steps Prepare tissue for transformation • Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos Introduce DNA • Agrobacterium or gene gun Culture plant tissue • Develop shoots • Root the shoots Field test the plants • Multiple sites, multiple years
- 35. The Lab Steps
- 36. Lab Testing The Transgenics Insect Resistance Cold Tolerance Transgene= Transgene= Bt-toxin protein CBF transcription factors
- 37. The Next Test Is The Field Herbicide Resistance Non-transgenics Transgenics
- 38. Final Test Consumer Acceptance RoundUp Ready Corn Before After
- 39. The Organization of the CBF Transcriptional Activator Protein Encoding Genes in Tomato
- 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. Cold acclimation and freezing tolerance in Arabidopsis thaliana 2 days -5oC; 4 days recovery at 20oC No acclimation 4 days ~ 2 oC
- 42. Arabidopsis Genes Cold – regulated genes - COR - 1st identified low temp. induced genes
- 43. Function of COR15a Two 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. COR Genes Promoter elements – C-repeat/Drought responsive element (CRT/DRE)
- 45. Promoter elements of COR Genes: – C-repeat/Drought responsive element (CRT/DRE) COR genes was accomplished by overexpressing the Arabidopsis transcriptional activator CBF1 (CRT/DRE binding factor 1) CBF1 binds to CRT/DRE DNA regulatory element present in the promoters of the COR genes CBF1 resulted in a greater increase in freezing tolerance than did expressing COR15a alone.
- 46. CBF1 pathway might control one set of cold-acclimation response CBF1 has a mass of 24 kDa, has AP2 domain in Stockinger: Arabidopsis, tobacco, and other plants proteins. have demonstrated that Ap2 domain includes a Ohme-takagi DNA-binding region. CBF1is a transcriptional activator that can activate CRT/DRE-containing genes and was a probable regulator of COR gene expression in Arabidposis CBF1 appears to be an important regulator of the cold- acclimation response, controlling the level of COR gene expression.
- 47. CRT/DRE Sequences COR15a ATTTCATGGCCGACCTGCTTTTT ACTTGTTGGCCGACATACATTTG CAAAATAAACCGACAAGGTTGCA COR15b ACTTGATGGCCGACCTCTTTTTT TGTGGCATACCGACTTCTAGATG COR78 AAGATCAAGCCGACACAGACACG GATATACTACCGACATGAGTTCC AATATCATACCGACATCAGTTTG AGACATGGACCGACTACTAATAA RD29b AAACGTGGACCGACTAAAACTAA KIN1 AAATAGCTACCGACATAAGGCAA ACTACTGATCCGACATCAAAACC COR6.6 AAAAAGCTACCGACATAAGCCAA COR47 ATTCATCTACCGACTTCAAGAAA CAATCAAAGCCGACCATTCAGCT CCCACATGACCGACATCTTATGC TAGCTTTAGCCGACGTGTCTAAT ERD10/LTI45 ACCGACCGACCGACGTAAAAGAA ATTCATCCACCGACCGACCGACG
- 48. CBF Gene Family Binds CRT/DRE element Transcription activators Plays a regulatory role – Overexpression induces COR genes
- 49. CBF Gene Family CBF is a member of a small gene family encoding three closely related transcriptional activator. CBF1, CBF2 and CBF3 are physically linked n direct repeat on chromosome 4 near molecular markers PG11 and m600(-71cM) Like CBF1, both CBF2 and CBF3 proteins can activate expression of reporter genes in yeast that contain the CRT/DRE as an upstream activator sequence, indicating that these two family member are also transcriptional activators.
- 50. CBF1 1 32 44 47 106 213 NLS AP2 Domain Activation Domain “Zip-Code” to get protein into nucleus Binds to CRT/DRE “Flips the switch” Causing Gene Activation
- 51. COR Gene Activation by CBF Activation Domain DNA Binding Domain COR GENE CR CR CR TA TA T/ T/ T/ DR DR DR E E E
- 52. Thomashow 2001
- 53. Proposed Regulatory Mechanism • Warm temperature COR
- 54. Proposed Regulatory Mechanism • Cold temperature CBF CBF COR
- 55. Proposed Regulatory Mechanism • Cold temperature CBF CBF COR
- 56. Project Overview Most plants possess CBFs – Based on BLASTs of different crop species Tomato – Cultivated species, TA491 – Cold tolerance wild species, LA407
- 57. Chromosomal Location of the 6 Arabidopsis CBFs At1g12610 DREB1F CBF4 DREB1D At5g51990 At1g63030 CBF1 CBF3 CBF2 DREB1E DREB1B DREB1A DREB1C At4g25490 At4g25480 At4g25470 1 4 5
- 58. CBF Signature Sequences AtCBF1: PKKPAGRKKFRETRHP FADSAWR AtCBF2: PKKPAGRKKFRETRHP FADSAWR AtCBF3: PKKPAGRKKFRETRHP FADSAWR AtCBF4: PKKRAGRKKFRETRHP FADSAWR AtCBF5: PKKRAGRRIFKETRHP FSDSAWR AtCBF6: PKKRAGRRVFKETRHP FADSAWR LeCBF1: PKKPAGRKKFRETRHP FSDSAWR LeCBF2: PKKPAGRKKFRETRHP FADSVWR GmCBF1: PKKRAGRKKFRETRHP FADSAWR GmCBF2: PKKRAGRKKFRETRHP FADSASR GmCBF3: PKKRAGRRVFKETRHP FADSRWR MtCBF3: PKKRAGRKKFKETRHP FADSAWR MtCBF2: PKKRAGRKKFKETRHP FADSAWR MtCBF1: PKKRAGRRVFKETRHP FADSAWR HvCBF1: PKRPAGRTKFHETRHP FADSAWR HvCBF3: PAKRPAGRTKFRETRHP FADSAWL Consensus: PKKPAGRKKFRETRHP FADSAWR R Rx K S
- 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. Lambda Phage Clone Phage Genomic clone: Le3
- 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. Sequence analysis: • Alignment sequence data using Sequencher software • Detect the CBF loci from the sequence, find the open reading frame • Protein sequence alignment of AtCBF and LeCBF
- 63. 23/CHENG4/M13R.phd.1 21/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.1 1 188 363 585 866 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
- 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. Protein sequence alignment of LeCBF1.2.3 LeCBF1 MNIFETYYSDSLILTESSSSSSSS--------SFSEEEVILASNNPKKPAGRKKFRETRH 52 LeCBF2 MDIFESYYSNSFVESLLSSSLSISDTNNLNHYSPNEEVIILASNNPKKPAGRKKFRETRH 60 LeCBF3 -----MFYSDPRIESCSSFSDSIR-------ANHSDEEVILASNNPKKPAGRKKFRETRH 48 :**:. : : * * * . .:* :********************* AP2 Domain LeCBF1 PIYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAALALRGRSACLNFSD 112 LeCBF2 PVYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 120 LeCBF3 PVYRGVRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 108 *:***:****************************************:***********:* LeCBF1 SAWRLPIPASSNSKDIQKAAAQAVEIFRSEEVSGESPETSENVQESSD--FVDEEAIFFM 170 LeCBF2 SVWRLPIPASSNSKDIQKAAAEAAEIFRSEEVSGESPETSENVQESSD--FVDEEALFSM 178 LeCBF3 SAWRLPTPDSSDTKDIQKAAAQAAEIFRPLKSEEEESVVKDQSTTPDDMFFMDEEALFCM 168 *.**** * **::********:*.****. : . *.. ..:: ..* *:****:* * LeCBF1 PGLLANMAEGLMLPPPQCAEMGDHCVETD---AYMITLWNYSI 210 LeCBF2 PGLLANMAEGLMLPPPQCLEIGDHYVELADVHAYMP-LWNYSI 220 LeCBF3 PGLLTNMAEGLMVPPPQCTEMGDHVEADD-----MP-LWSYSI 205 ****:*******:***** *:*** * **.***
- 67. Protein sequence alignment of AtCBF and LeCBF AtCBF4 MNPFYSTFPDSFLSI-SDHRSPVS--------DSSECSPKLASSCPKKRAGRKKFRETRH 51 AtCBF5 MNPFYSTFPDSFLSI-SDHRSPVS--------DSSECSPKLASSCPKKRAGRKKFRETRH 51 AtCBF1 MNSF-SAFSEMFG---SDYEP-----------QGGDYCPTLATSCPKKPAGRKKFRETRH 45 AtCBF2 MNSF-SAFSEMFG---SDYESPVS--------SGGDYSPKLATSCPKKPAGRKKFRETRH 48 AtCBF3 MNSF-SAFSEMFG---SDYESSVS--------SGGDYIPTLASSCPKKPAGRKKFRETRH 48 LeCBF1 MNIFETYYSDSLILTESSSSSSSS--------SFSEEEVILASNNPKKPAGRKKFRETRH 52 LeCBF2 MDIFESYYSNSFVESLLSSSLSISDTNNLNHYSPNEEVIILASNNPKKPAGRKKFRETRH 60 LeCBF3 -----MFYSDPRIESCSSFSDSIR-------ANHSDEEVILASNNPKKPAGRKKFRETRH 48 AtCBF6 ---------------------------------MNNDDIILAEMRPKKRAGRRVFKETRH 27 .: ** *** ***: *:**** AtCBF4 PIYRGVRQRNSGKWVCEVREPNKKSRIWLGTFPTVEMAARAHDVAALALRGRSACLNFAD 111 AtCBF5 PIYRGVRQRNSGKWVCEVREPNKKSRIWLGTFPTVEMAARAHDVAALALRGRSACLNFAD 111 AtCBF1 PIYRGVRQRNSGKWVSEVREPNKKTRIWLGTFQTAEMAARAHDVAALALRGRSACLNFAD 105 AtCBF2 PIYRGVRQRNSGKWVCELREPNKKTRIWLGTFQTAEMAARAHDVAAIALRGRSACLNFAD 108 AtCBF3 PIYRGVRRRNSGKWVCEVREPNKKTRIWLGTFQTAEMAARAHDVAALALRGRSACLNFAD 108 LeCBF1 PIYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAALALRGRSACLNFSD 112 LeCBF2 PVYRGIRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 120 LeCBF3 PVYRGVRKRNSGKWVCEVREPNKKTRIWLGTFPTAEMAARAHDVAAIALRGRSACLNFAD 108 AtCBF6 PVYRGIRRRNGDKWVCEVREPTHQRRIWLGTYPTADMAARAHDVAVLALRGRSACLNFAD 87 *:***:*:**..***.*:***.:: ******: *.:*********.:***********:*
- 68. AtCBF4 SAWRLRIPETTCPKEIQKAASEAAMAFQNETTT---EGS-KTAAEAEEAAGEGVREGERR 167 AtCBF5 SAWRLRIPETTCPKEIQKAASEAAMAFQNETTT---EGS-KTAAEAEEAAGEGVREGERR 167 AtCBF1 SAWRLRIPESTCAKDIQKAAAEAALAFQDETCD---TTTTDHGLDMEETMVEAIYTPE-- 160 AtCBF2 SAWRLRIPESTCAKEIQKAAAEAALNFQDEMCH---MTTDAHGLDMEETLVEAIYTPE-- 163 AtCBF3 SAWRLRIPESTCAKDIQKAAAEAALAFQDEMCD---ATT-DHGFDMEETLVEAIYTAE-- 162 LeCBF1 SAWRLPIPASSNSKDIQKAAAQAVEIFRSEEVS---GESPETSENVQE------------ 157 LeCBF2 SVWRLPIPASSNSKDIQKAAAEAAEIFRSEEVS---GESPETSENVQE------------ 165 LeCBF3 SAWRLPTPDSSDTKDIQKAAAQAAEIFRPLKSE---EEESVVKDQSTT------------ 153 AtCBF6 SAWRLPVPESNDPDVIRRVAAEAAEMFRPVDLESGITVLPCAGDDVDLGFGSGSGSGSGS 147 *.*** * :. .. *::.*::*. *: : AtCBF4 AEEQNGGVFYMDDEALLGMPNFFENMAEGMLLPPPE---VGWNHN-DFDGVGD----VSL 219 AtCBF5 AEEQNGGVFYMDDEALLGMPNFFENMAEGMLLPPPE---VGWNHN-DFDGVGD----VSL 219 AtCBF1 ---QSEGAFYMDEETMFGMPTLLDNMAEGMLLPPPS---VQWNHNYDGEGDGD----VSL 210 AtCBF2 ---QSQDAFYMDEEAMLGMSSLLDNMAEGMLLPSPS---VQWNYNFDVEGDDD----VSL 213 AtCBF3 ---QSENAFYMHDEAMFEMPSLLANMAEGMLLPLPS---VQWNHNHEVDGDDDD---VSL 213 LeCBF1 ----SSD--FVDEEAIFFMPGLLANMAEGLMLPPPQ---CAEMGDHCVETD---AYMITL 205 LeCBF2 ----SSD--FVDEEALFSMPGLLANMAEGLMLPPPQ---CLEIGDHYVELADVHAYMP-L 215 LeCBF3 ----PDDMFFMDEEALFCMPGLLTNMAEGLMVPPPQ---CTEMGDHVEADD-----MP-L 200 AtCBF6 EERNSSSYGFGDYEEVS---TTMMRLAEGPLMSPPRSYMEDMTPTNVYTEEEMCYEDMSL 204 . : . * : : .:*** ::. * *
- 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’ LeCBF2 3’ 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’ LeCBF1 3’ LeCBF2 3’ LeCBF3 eIF4A
- 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. Thank You!