How Transgenic plant is used in How Transgenic plant is used in Agricultural FieldAgricultural Field

转基因植物在农业生产中的应用

chenghm@caas.net.cn，Tel：82106125, Crop institue buliding 401

Dr. Hongmei Cheng程红梅

Biotechnology Research Institute, CAAS

Biotechnology Research Institute was founded in 1986

BRI-CAAS

Permanent staff: 112Academician: 1Professor: 26Associate Professor: 32Assistant Professor: 24PhD and Master Students>150

Organizations-Departments and labs

Research Departments: 2Department of Plant Biotechnology and Molecular BiologyDepartment of Molecular Microbiology

Laboratories: 8Laboratory of Plant Genetic EngineeringLaboratory of molecular biology for plant stress toleranceLaboratory of plant metabolic engineering Laboratory 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

Research Centers: 1. Research Center for Crop Molecular Designing2. Research Center for Microorganism Genetic Engineering

3. Research Center for Biosafety Assessment of GMOs4. Key laboratory for Crop Molecular Biology, Ministry of

AgricultureEngineering Center1. Center for Biotechnology Products

Organizations-Research Centers

Remarkable Achievements

Insect Resistance

Transgenic Bt cotton resistant to ball worm

1. Commercialized since 19982. In 2007, more than 70% cotton

are transgenic, Accumulated acreage: 2.4 million ha.

3. Accumulated benefit since 1998: >126 billion RMB

Without signal peptide With signal peptide targeting to extracellular space

Without signal peptide With signal peptide targeting to extracellular space

Efficiency corn transformation system

Phytase corn in greenhouse

Phytase Expression Vectors

Molecular Screen and enzymatic activity assay

Molecular Farming and Bioreactor

Phytase corn in field

1. Used as feed additive to increase the efficiency use of phosphorus and proteins\metal ions bioavailability

2. Highly expressed lines have been obtained

3. Biosafety assessment of transgenic corn completed

Producing phytase by transgenic corn

What is Biotechnology?

How about some definitions

General DefinitionThe application of technology to improvea biological organism

Detailed DefinitionThe application of the technology to modify thebiological function of an organism by adding genesfrom another organism

But we know nature does not have all of the traits we need

• Here we see bean has manyseedcoat colors and patternsin nature

•Nature has a rich source of variation

These definitions imply biotechnologyis needed because:

But nature does not contain all thegenetic variation man desires

•Fruits with vaccines

•Grains with improved nutrition

Central Dogma of Molecular Genetics

(The guiding principle that controls trait expression)

DNA(gene)

RNA

Protein Trait(or phenotype)

Transcription

Translation

Plant height

Seed shape

In General, Plant Biotechnology TechniquesFall Into Two Classes

• Identify a gene from another species which controlsa trait of interest

• Or modify an existing gene (create a new allele)

Gene Manipulation

• Introduces that gene into an organism• Technique called transformation• Forms transgenic organisms

Gene Introduction

Genes Are Cloned Based On:

Similarity to known genes

Homology cloning (mouse clone used to obtain human gene)

Protein sequenceComplementary genetics (predicting gene sequence

from protein)

Chromosomal locationMap-based cloning (using genetic approach)

Human clonelibrary

Clones transferredto filter

Mouse probeadded to filter

Hot-spots are humanhomologs to mouse gene

Homology Cloning

Complementary Genetics

1. Protein sequence is related to gene sequence NH3+-Met-Asp-Gly--------------Trp-Ser-Lys-COO-

ATG GAT-GCT TGG-AGT-AAAC C C G

A TCTG C

AG

2. The genetic code information is used to design PCR primersForward 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 nucleotidesReverse primer is the reverse complement of the gene sequence

3. Use PCR to amplify gene fragment

Complementary Genetics(cont.)

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’

3’ 5’

5’ 3’

c. two copies of the template DNA made (72C)

Human clonelibrary

Clones transferredto filter

PCR fragmentprobe added to filter

Hot-spots are human geneof interest

Complementary Genetics(cont.)

4. Gene fragment used to screen library

Map-based Cloning

1. Use genetic techniques to find marker near gene

Gene Marker

2. Find cosegregating markerGene/Marker

3. Discover overlapping clones(or contig) that contains the marker Gene/Marker

4. Find ORFs on contigGene/Marker

5. Prove one ORF is the gene bytransformation or mutant analysis

Mutant + ORF = Wild type?Yes? ORF = Gene

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 enzymeIncreased Vitamin A content

Vitamin A biosynthetic pathway enzymes

Introducing the Gene orDeveloping Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

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

Gene of Interest

Coding Region• Encodes protein product

ex.: EPSP-carotene genes

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)

Promoter Coding RegionTP

Transit Peptide• Targets protein to correct organelle

ex.: RbCS (RUBISCO small subunit; choloroplast target

Selectable Marker

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]

Promoter Region• Normally constitutive

ex.: CaMV35s (Cauliflower Mosaic Virus 35S RNA promoter

Promoter Coding Region

Effect of Selectable Marker

Transgenic = Has Kan or Bar Gene

Plant grows in presenceof selective compound

Plant dies in presenceof selective compound

Non-transgenic = Lacks Kan or Bar Gene

X

Insertion Sequences

• Used for Agrobacterium-transformationex.: Right and Left borders of T-DNA

Required for proper gene insertions

TL TR

Let’s Build A Complex Cassette

pB19hpc (Golden Rice Cassette)

TL TRaphIV 35S Gt1 psy 35S rbcS crtl

HygromycinResistance

PhytoeneSynthase

PhytoeneDesaturase

T-DNABorder

T-DNABorder

SelectableMarker

Gene ofInterest

Gene ofInterest

InsertionSequence

InsertionSequence

• Transformation cassettes are developed in the lab

• They are then introduced into a plant

• Two major delivery methods

Delivering the Geneto the Plant

• Agrobacterium

• Gene GunTissue culturerequired to generatetransgenic plants

Plant Tissue CultureA Requirement for Transgenic Development

A plant part Is cultured

Callusgrows

Shootsdevelop Shoots are rooted;

plant grows to maturity

AgrobacteriumA natural DNA delivery system

• A plant pathogen found in nature

• Hormone genes expressed and galls form at infection site

• Delivers DNA that encodes for plant hormones

• Infects many plant species

Gall onstem

Gall onleaf

• DNA incorporates into plant chromosome

But Nature’s AgrobacteriumHas Problems

Infected tissues cannot be regenerated (via tissue culture)into new plants

Transferred DNA (T-DNA) modified by• Removing phytohormone genes

• Retaining essential transfer sequences

• Adding cloning site for gene of interest

• Phytohormone balance incorrect regeneration

Solution?

Why?

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 andincorporates into chromosome

• Integration process unknown

Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue• Develop shoots• Root the shoots

Field test the plants

• Leaf, germinating seed, immature embryos• Tissue must be capable of developing into normal plants

• Agrobacterium or gene gun

• Multiple sites, multiple years

The Lab Steps

Lab Testing The Transgenics

Insect Resistance

Transgene=Bt-toxin protein

Cold Tolerance

Transgene=CBF transcription factors

The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

Final TestConsumer Acceptance

RoundUp Ready Corn

Before After

The Organization of the CBF Transcriptional Activator Protein Encoding Genes in Tomato

Plant Responses to ColdPlant 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

Cold acclimation and freezing tolerance in Arabidopsis thaliana

No acclimation 4 days ~ 2 oC

2 days -5oC; 4 days recovery at 20oC

ArabidopsisArabidopsis GenesGenes

Cold – regulated genes- COR

- 1st identified low temp. induced genes

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

COR GenesCOR Genes

Promoter elements– C-repeat/Drought responsive element

(CRT/DRE)

Promoter elements of COR Genes: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.

CBF1 pathway might control one set of cold-acclimation response

Stockinger: CBF1 has a mass of 24 kDa, has AP2 domain in Arabidopsis, tobacco, and other plants proteins.

Ohme-takagi have demonstrated that Ap2 domain includes a 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.

CRT/DRE Sequences

ATTCATCTACCGACTTCAAGAAACAATCAAAGCCGACCATTCAGCTCCCACATGACCGACATCTTATGCTAGCTTTAGCCGACGTGTCTAAT

COR47

ATTTCATGGCCGACCTGCTTTTTACTTGTTGGCCGACATACATTTGCAAAATAAACCGACAAGGTTGCAACTTGATGGCCGACCTCTTTTTTTGTGGCATACCGACTTCTAGATG

COR15a

COR15b

AAGATCAAGCCGACACAGACACGGATATACTACCGACATGAGTTCCAATATCATACCGACATCAGTTTGAGACATGGACCGACTACTAATAAAAACGTGGACCGACTAAAACTAA

COR78

RD29bAAATAGCTACCGACATAAGGCAAACTACTGATCCGACATCAAAACCAAAAAGCTACCGACATAAGCCAA

KIN1

COR6.6

ERD10/LTI45 ATTCATCCACCGACCGACCGACGACCGACCGACCGACGTAAAAGAA

CBF Gene FamilyCBF Gene Family

Binds CRT/DRE elementTranscription activatorsPlays a regulatory role

– Overexpression induces COR genes

CBF Gene FamilyCBF Gene FamilyCBF 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 familymember are also transcriptional activators.

1 32 44 47 106 213

CBF1

“Flips the switch”Causing Gene Activation

Binds to CRT/DRE

“Zip-Code”to get proteininto nucleus

NLS AP2 Domain Activation Domain

Activation Domain

DNA Binding Domain

COR Gene Activation by CBF

CRT/DRECRT/DRE

CRT/DRE

TATA

COR GENE

Thomashow 2001

Proposed Regulatory Proposed Regulatory MechanismMechanism

COR

• Warm temperature

Proposed Regulatory Proposed Regulatory MechanismMechanism

• Cold temperature

CORCBFCBF

Proposed Regulatory Proposed Regulatory MechanismMechanism

• Cold temperature

CORCBFCBF

Project OverviewProject Overview

Most plants possess CBFs– Based on BLASTs of different crop species

Tomato– Cultivated species, TA491– Cold tolerance wild species, LA407

51

Chromosomal Location of the 6 Arabidopsis CBFs

At1g12610DREB1F CBF4

DREB1DAt5g51990

4

CBF1DREB1B

At4g25490

CBF3DREB1A

At4g25480

CBF2DREB1C

At4g25470

At1g63030DREB1E

AtCBF1: PKKPAGRKKFRETRHP FADSAWRAtCBF2: PKKPAGRKKFRETRHP FADSAWR AtCBF3: PKKPAGRKKFRETRHP FADSAWRAtCBF4: PKKRAGRKKFRETRHP FADSAWRAtCBF5: PKKRAGRRIFKETRHP FSDSAWRAtCBF6: PKKRAGRRVFKETRHP FADSAWR

LeCBF1: PKKPAGRKKFRETRHP FSDSAWRLeCBF2: PKKPAGRKKFRETRHP FADSVWR

GmCBF1: PKKRAGRKKFRETRHP FADSAWRGmCBF2: PKKRAGRKKFRETRHP FADSASRGmCBF3: PKKRAGRRVFKETRHP FADSRWR

MtCBF3: PKKRAGRKKFKETRHP FADSAWRMtCBF2: PKKRAGRKKFKETRHP FADSAWRMtCBF1: PKKRAGRRVFKETRHP FADSAWR

HvCBF1: PKRPAGRTKFHETRHP FADSAWRHvCBF3: PAKRPAGRTKFRETRHP FADSAWL

Consensus: PKKPAGRKKFRETRHP FADSAWRR Rx K S

CBF Signature Sequences

ObjectivesObjectives• 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

Phage Genomic clone:

Le3

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

Lambda Phage Clone

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

23/CHENG4/M13R.phd.121/CHENG3/M13R.phd.1

03/cheng3/E10.phd.143/CHENG3/ES173.phd.1

46/CHENG8/ES176.phd.144/CHENG3/ES204.phd.1

30/CHENG8/M13F.phd.131/CHENG3/ES184.phd.114/CHENG3/ES155.phd.1

31/CHENG3/E7.phd.143/CHENG3/ES203.phd.1

29/CHENG7/M13R.phd.130/CHENG3/ES183.phd.1

01/cheng3/E8.phd.128/CHENG7/M13F.phd.1

42/CHENG3/ES172.phd.126/CHENG6/M13F.phd.1

15/CHENG3/ES156.phd.122/CHENG4/M13F.phd.1

20/CHENG3/M13F.phd.144/CHENG6/ES174.phd.1

07_CHENG2_E2322/CHEN2/M13F.phd.1

34/CHENG6/ES187.phd.102/cheng2/E9.phd.1

19/CHENG2/ES163.phd.106_CHENG2_E22

28/CHENG2/ES181.phd.127/CHENG2/E3.phd.1

05/cheng2/E12.phd.1

1 4,905188 1,585 2,183 2,775 3,686585 2,446 3,953363 866 1,038 1,234 1,939 3,021 3,349 4,241 4,506

CCAAAAGGGAAGTATCAAAGTACAGAAAAAAACTAAAAATATGCCAAGTTAGACGCACGGAAGATTTGGAAGTTGAAACTTAACTTTTCTTAAACCCACAGCCCCACTCCAGCTGTCATATAAAACAGCTGCCCCACTCTATTTTTTAATAACAGCCTGTCTACTTATCACCACCCTCTAACTCCGTGTTCTTTGGTCTCAACTATATATAGAAATCAAACTTTTCACATTTTACCATAACAATTAAACTCTCTAACATCATAAATATCACTAGTTAAAGAAAGAAACAAAAATATAAATCGATATGTTTTATTCGGACCCACGTATAGAATCTTGTTCATCGTTTTCTGACAGTATTAGAGCCAATCATTCTGACGAGGAAGTTATTTTAGCTTCAAATAATCCGAAGAAGCCAGCTGGCAGAAAGAAGTTTCGAGAAACTCGACATCCAGTGTACAGGGGAGTGAGGAAGAGGAATTCTGGAAAATGGGTTTGTGAAGTCAGAGAACCAAATAAGAAGACGAGGATTTGGCTTGGTACTTTTCCTACTGCTGAAATGGCGGCTAGAGCTCATGATGTGGCGGCTATAGCATTAAGAGGACGTTCAGCTTGTTTGAATTTTGCTGACTCTGCTTGGAGGCTGCCTACTCCAGATTCCTCTGACACTAAGGATATTCAAAAGGCGGCCGCTCAGGCCGCCGAAATCTTCCGACCTTTAAAGTCGGAGGAAGAAGAATCAGTGGTTAAAGATCAATCTACTACTCCAGATGATATGTTTTTTATGGATGAGGAAGCGTTATTCTGCATGCCGGGTTTACTTACGAATATGGCGGAAGGATTAATGGTACCTCCACCTCAATGTACTGAAATGGGAGATCATGTGGAAGCTGATGATATGCCTTTATGGAGCTATTCTATATAATAAGTAAGTATAATGAGAGGAGTAACAATGCTAAGAGTGAAGTTTATTAGTTTCGTGCTTAATATTTGGATATGGTACGAATTAGTGTATAAGTATTGTAATTTGTAATGATCATGTAGATATTACTAGTATTGCTATATACTATTATAACAAAATGGTTGAAGCTAAATGAGAATCATTGGCGTATATAAGACTATTGTGTGTTTTATGACAGTTAGTCTTAGAGTTTTTTCTCATGGTTGAATTTGGTTAAGAAGCTGTTAAATGCGTTGTTCCACCAGCTTCGGAAAAACAACAGACACATACTCTAAAAAAAGCATAAAGCATTTGCTTCTGGTTTAAGCAACTGAGTGAAAAAGTAGATTTGTGGAGTATTTTTTCAAGCCGATTACTATGTCACAATCAATCAAAGAACATTGCTTATATCATAATTTTTATAAATTTTCAAAAATAAATATATCTATATATACATATAATTATTTTTTTAAAAGTTTAACATGTACACATGTTTCTCAATTTTACACGTGTGTCCGCCTATGATACAAATTTTATTAGTAATGACAATTGTAGAACTTTCTAGAATGAAATAACAATGGAGACAATTCAAATAGTTTGGAGATATATATATGTCTCAACATTGTGGTACTAATCCAATTCCAAGCATATCGATGCTGGAAATGATGCACGTGGTCCACGCGTATAATTTCCCGCGTGAGAAAATGAAAAGTAATTTATTGGAGTTGCAATAATTGATGATATAATTAACCGTCAAAAGCGTGTGTTGAGTTTTAATCAGTTATAAATTGGTACTTAGTTCACTTGTGACTTCATACATATACATATAATCATTTCAAAGGTCAATTTTCAAACTCATCTTTCAATTGGATCAAGTAGGGGGCGGCTATATATATATATATATATTGGCCTAAGAATAGAACGGCAACTTACCCTTCACCTTCCACTATCTTTTCAAAGATTCTCAATAATCAGTAGTATGATAATGAACAATGCTAAATGATCAACAAAATTTAATCAGAAATTTTAAAAAAATTGTAACGTCTCTTTTATTTTAATATAATTTTTTTTATTTTAATAAAAGAATAAAAATATAAAAAAATATCATTTTAATCAAATTCTGATCAAATTTGCTGACCATAAGAATTTTTTCATTTATTAATTAGTTTTATTCTTCATTATACTATCGTACTTATAAAAATCTCTTTCATTATGAAACTTTACATATTTACCTTTTATTTGAATAGATTATCCTAAAATTGGTCAAAAATATCTTTGTCATTATGGAACTCAAACTTCACTTTTAGGGTTGGGGTACTTATAAATATAATAGTTTGGAATAAAATTATAAATCTAATAAAATATACTTGGGTTTATATCTAGTCTCCTAAAATAGAAATAACACACACTCTCTCTCACACACACACACACTCCCCTTTTCATTCCCTTCATATTTTGTTACTCCTATTATTTTTAACTATTCTATTCTAGTCTAATTTCTCCTCTACAAAGCTTGAATCTCTAGATATAGTTATTGTCCTCAGTTATGTTATTTTCTCATTTCAAGTATTTTCAGCTACTTCCCAACATTAGAAAAGTCCATAAAATATAAATAATAATATATAAACATAAAATAAAATTAAAAATTTATTATATATAAAAAATAGTAATTTTTTTTTGGAATGAAACTTAACCAAACTCATAAAATATGCTAATTAATTAATAAGGGATATATAGGTAAATATGTATGTATGGAAGAGACATTTTAATCTTAAAAAAATAATTTTCTTCTCTGTTTCATTTTTTTAAGAAGCAGAACTTTTAGATTCTTCCCAACAACAGAACAACTGCTTCTTACTTTTTGCAAACACTTGATTTTTCAAAAAGAAAAAACATACTTTTTTCTAGGAAAAAAAAACGCTTTTGGCCTTCCAATGAATCCAATTCTAATTCAATCTTAACAAATTTAGGGTATAATCAGAAAAAAAAATATTTTTTCTTAATTTATTAAAAGTGACCAGTAAAAATGGAAATTAGATTAGAAAATATTTGTCGAATAAATAGAGACGAAGAGAGTTTAAAAAAGAAGTTGATGAATGCTGACCTTTTCCTTTGACAACTATTGGTTCAATGAATCTCCAAAGATTTATCTCTCAATTTTAAAAAATTGGTGATGACGAGATAGATGGTATAAAATAGATGCAACAAGAATAATTTTTTTTATTTTTTTTAATGTTATCATATTGAAATGACAAAGATTGGTCAGTATATATTCCAAAAAGGAAGTAAAGAGGAAAAGTTTTACAAGTCACAAGTTGCCACACGAGTTGTACGCAAATCCACTTGTCCCATAAAACAAAACAGCTGGGCTTACGCTTTTATAATCCAGCCTGTATCCTTTAATTATCACTCCGTGTTCTCTTCTCCTTTCACTATCATACTCTACTTTCCACTATAAATATATGTAACCAACACATAACACTTCTTTAACTCAACAATTATACAAATACTTTCTATTTTTAGCTCTCAACAACAATGAATATCTTTGAAACCTATTATTCAGACTCGTTAATTTTAACCGAATCATCTTCTTCTTCATCGTCATCGTCGTTTTCTGAAGAGGAAGTTATTTTAGCTTCGAATAACCCGAAAAAGCCAGCTGGCAGGAAGAAGTTTCGAGAAACACGGCATCCGATATACAGGGGAATCAGGAAGAGGAATTCAGGAAAATGGGTTTGTGAAGTCAGAGAACCAAATAAGAAGACAAGGATTTGGCTTGGTACTTTTCCTACGGCTGAAATGGCGGCTAGAGCTCATGACGTGGCGGCTTTAGCATTAAGAGGCCGTTCTGCTTGTTTGAATTTCTCTGATTCTGCTTGGAGGCTGCCTATCCCTGCTTCCTCCAACTCTAAAGATATTCAAAAGGCGGCCGCTCAGGCCGTCGAAATCTTCCGATCGGAAGAAGTTTCAGGAGAATCTCCTGAAACGTCAGAAAATGTGCAAGAGAGTAGTGACTTCGTGGATGAGGAGGCGATCTTTTTCATGCCAGGATTACTTGCAAATATGGCAGAAGGACTTATGCTACCTCCACCTCAATGTGCAGAAATGGGAGATCATTGTGTGGAAACTGATGCCTACATGATAACTTTATGGAATTATTCTATCTAAAATAGTAGTACAATTTATCAAATTACTAGGATTTAGAAGATTTTGTTAGTTTTTGGTATTCAGTATTTAGATACTAAGAATGTATATTATTAGTATTTTTATTTTGGCCAAATACATGAACATGAACAGAAACTTGTTGGGTTTTTTTACTCAGGTACCTCAACTACATCATTTTTCTATTGATTATTGAACTACACATAATTTGTTTCTTTAAAACACTGTTGGTTGATTTTGATCGACTTTTTTATTATAAATGTCTTCAATAATGTTCGAATTGTAATAATTTTGATTAAATGAATGAAGACAAACCGTGTTAATCTTAATTGTTTTCTAATGTGTTCAAATGACTTAAGTAAAACACAATTATTCTTGAACATTTTCACTATCAATTGGATTAATGAGTTGTGGAACAACATATCTATTCTCTATCAATAATCTTCACAAATCTGGTTCCACATCAGACAACAGTGTTTGTTTAAACAGAACAAATTATGGGGATTCAATGGTTCAATAGGAAAATGACGTAGTAAAGGAATCTGAAAATAAAAAAATCGAACAAATTTAGGGATCTGCTTATTGTACCGAACCATGTAGGTAGATAGTAGTGCCACCAAATAATGACACGTGTCAATGGGATGACTTGGTTTTGGCAGTAGTGAGAAGTAAAGATTAGCGTTGCAAATTTCAAGCCGTCATATTTGAATAAATGAAGTGTGGAGTGATATGACAATGTTCAATATTTTTTGCCATTCCGAGTATTGAAGAATTACAATTTCTAACTTATTTTTCGTAATTACTGAGTATCTAAATGTTAATTTTATGAATCCAATCTAAGCAAAGTTATCTGATATTGAAAAAACTTGTTTACTTAAAAACTAAGAAAACTAAAAATATATAATCCCTTCGTTCAAAAATAATGAAGGGGTGCTCAATATGAGTCAACCATATTCAAATTAAAGTTTCAATTTCAATTCCAAAGTTATTTGTCTCAACATTGAAAAATTTCAGTATTATGAAATTACAAAATAAATAAGATATAACTTTTTCATGTTTATAAACACTTTAAAACTGTAATATATAAAATATGAGTAATGAGTAGAGTGAAATATAGGAAAGTTTCCAAATATAGCTTTTAGCCTATCGTTATCTATCTAGAATGCCATTTATTGTACTACTGCCTCCTCTTTGTACGCACTATTTTGACTTGTTCTTTTCCTTCATTCGTGTACAATTTTATTTTTCCACAAAGTTTTCGTAGGTTTAGGTTAAGATAGTTAGAATTTCTTATAAATTATTTAGTTTCTTGATTCTAATTTAAGTAACACAGTTCTAATAAATACTATACGAAGTATTATAAAATAAAAAAATAAAATTTATGATTTAAAACATAATATTTGTGTGACTATAGAAGTTATGTTAGTAAATAAGTATAACATTAGTTTCTATTGGTGAATATAACAAGCAATTATTTTAGGGACAGATTAACAATGCATTCCATGTCTAAGTCCAATTCTTTTTTCCCAATAATACTTATTTCCTTTAATTTTAAAAAAAATCTCCTCTTTTTATTCTGTTTAAAAAAAATATGATTTTTTTTTTGCTAGCAACTTTTCACGTGACATGTTTAAGGCCATAATATTAAAGTGTAGTTTTATACATTTGACATAACTTTAAATTAACACTACATGATCAAAAAAATTATTTTTTAAAACTTCGTGTCAAGTTAAACTAAACCAATTTTTATAAAACGGATGAAGTATTAAATTAGATGCACACTTTATTAATCACGTGAATATAACTAGCCTAATGAGCAAGAAGACTTGTTGAAGTCAATATATATTTCATGTGGACCTTAGACAAAAATAGTTTATTACTCTTTTATATTTCAATTTACGAAATCTTAAAATTTAATATATTTGTAAACAATATACAAAAATACTTATAAGTTATAACAATTAATATTTTAAAAATATTTAAAATATAAAATTTAATAATCAAAAATATATTTATTTAAATTTTAAAATTAAAAATATATCACGTATATTGAGATCGAGAACCCAGTACAAATATTGTAGATGAGATCTATTCCTTTTAGTTAGGAAGGAAGGAAGAAGGATAGGCAAAAAGTAGAAAGTTTGCCACATCAGCAGAAAGGCTACACGAATTATACACACTTGAGACTATAAAACAGCTGTCTACTTATCACTATCCAACTCCGTGTAATACCGAACTTTTTTAAATTCAACACTTCACTTATCATATGTTTTATATATATGCATTGAGAAAATCCAATTTCATAATTCACCACAAACCCAAAAACGTCCATCCATCGTACACTACTATATTTTACTCTCTCGTCAAAATAGTATTATCATATCATGGATATCTTTGAATCCTATTATTCAAATTCTTTCGTTGAATCATTATTATCATCGTCATTATCAATATCTGATACTAATAATCTCAATCACTACTCCCCTAATGAGGAAGTTATTATTTTAGCTTCGAATAACCCGAAAAAGCCAGCTGGCAGGAAGAAGTTTCGAGAAACTCGACATCCAGTATACAGGGGAATCAGGAAGAGGAATTCAGGAAAATGGGTTTGTGAAGTCAGAGAACCAAATAAGAAGACAAGGATTTGGCTTGGTACTTTTCCTACGGCTGAAATGGCGGCTAGAGCTCATGACGTGGCGGCTATAGCATTAAGAGGCCGTTCTGCTTGTTTGAATTTCGCTGATTCAGTTTGGAGGTTGCCTATACCTGCTTCCTCCAACTCTAAAGATATTCAAAAGGCGGCCGCTGAGGCCGCCGAAATCTTTCGATCGGAAGAAGTTTCAGGAGAATCTCCTGAAACGTCAGAAAATGTGCAAGAGAGTAGTGACTTCGTGGATGAGGAAGCGCTGTTTTCCATGCCAGGATTACTTGCAAATATGGCAGAAGGACTCATGCTACCTCCTCCCCAATGTTTAGAGATCGGAGACCATTACGTTGAATTAGCTGATGTGCACGCTTATATGCCTTTATGGAATTATTCTATATAATTACAAGATTTTAATAGTCAGTATTTTAATGGTACAAATCATGTATAGGTAAATGCGTAGTAACAATATTTGAATGAAACAAAGTGAACAAGTTCATCTAATTTATCAACAGCTATATTTCATCTATCCTTAGAATTTTCAATATATTTTATATTTTGTAGATTTAGAATTAATAACGTATAACTAATATATTGTGGTTCTTAGACTGTATTGAATAAGCGGGGAGGACATTGTCAATATGAAGAGTTTGTGGAATTTATTTTCATTATTTGTTATTTTTTTTATTTTAAAGGGAAAAGACATAAAAAGAAATTTTGAATTTGGTTCGAAAACTAACTTTAGTATTTTAACTATACGGGCGTTTAAATATTTCTCTTAACATCTTCAAAATGAATTAAAAACCACCCTGAGATTACTATTCCTTTCTCACTTGTCACATCATAGCCATGTAAGTGCCACAACAACATATCAGTATCATGTCGTTAGTTATTTTCATCATTTTTCTTTAGCATTTCTTTAAAATTAATTTACACTAATTAATTAATTCAAATTGCATTTTCTAAAATTAAAATTAAAAATGTCGACACATTTTTATTTTACCCCGTACCCAACTCCCACCCACCCCCTTACTTCTTTCTTCTTCCTTCTTCTTCACCATTTCTAACTCTCAATGTTCATTTTCCCCTCTCCATATTTTTCAACTACTTCCACCATCCAAACTCCTTTCATCGTAACACTCAAATCACCGCCGAAGCTCCATTATATATATATATATATATATTAGAAGATAAACATATAGAAACATGTAGTTAAAGGAAAGAAATAAAAACAATTTACTTCTAAGAAGACGA

Right LeftLeCBF3 LeCBF1 LeCBF2

NotI NotI NotI

4.4kb 3.2kb 3.1kb 8kb

Tomato Genomic Clone Le-3(~19kb)

CBF2CBF3CBF1

2.7kb 2kb

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

****:*******:***** *:*** * **.***

Protein sequence alignment of LeCBF1.2.3Protein sequence alignment of LeCBF1.2.3

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

*:***:*:**..***.*:***.:: ******: *.:*********.:***********:*

Protein sequence alignment of Protein sequence alignment of AtCBFAtCBF and and LeCBFLeCBF

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

. : . * : : .:*** ::. * *

3’ LeCBF2

3’ LeCBF1.

Drought Nacl(250mm)McM 1 2 4 6 8 24 1 2 4 8 16 24 1 2 4 8 16 24 (hours)

ABA(100µm)

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’ LeCBF2

3’ LeCBF1

eIF4A

3’ LeCBF3

3’ LeCBF3

3’ LeCBF2

3’ LeCBF1.

Drought Nacl(250mm)McM 1 2 4 6 8 24 1 2 4 8 16 24 1 2 4 8 16 24 (hours)

ABA(100µm)

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’ LeCBF2

3’ LeCBF1

eLF4A

Tomato CBF Genes Expression Pattern Under Different StressThe expression of LeCBF1 and LeCBF2 under various stress treatments was investigatedusing RNA Gel Blot analysis LeCBF1 but not LeCBF2 was found to be cold-responsive

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