Biological Function of RMR2 in Maize: Genetic Study through Fluorescent Tagging of RMR2 Protein

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Biological Function of RMR2 in Maize: Genetic Study

through Fluorescent Tagging of RMR2 Protein : Work In

Progress

Presented

By

Han Li

Immediate Supervisor:

Dr. Jacque Keele & Dr. Anding Luo

Lab PI:

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Outline

• Introduction

• Experimental outline and procedures

• Experimental results

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Central Dogma of Biology

• DNA

(deoxyribonucleic

acid)- genetic code

• Transcription-DNAmRNA

• Translation-mRNA protein

This is followed by Mendelian

inheritance

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New Discoveries in genetics

• Silencing

• Epigenetic regulation- heritable

changes in gene function without a

change in DNA sequence

Genes can be regulated by:

• Paramutation

– An interaction between alleles of

genes that leads to heritable changes

in gene expression

• DNA Methylation

• Histone acetylation/deacetylation &

methylation

• TGS and PTGS (Transcriptional

Genomic Silencing and Post

Transcriptional Genomic Silencing,

Plants)

• dsRNA (RNAi)

-Chandler, Vicki L. Cells (2007). 128: 641-645

-Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th_{. 2000.}

-McGinnis KM et al., (2006) Genetics, 173:1637-1647.

B-I*

B’

B-I*

B-I/B-I B-I*/B’

B’

B-I*/B’

B’/B’

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Role of RMR Protein

• RMR2 (required to maintain repression 2)

– Gene silencing the production of pigments

– Epigenetics studies

• Transcriptional gene silencing

– Regulates RNA polymerase

– DNA methylation

– Mutation are defective in paramutations

• RMR1

– Similar to RMR2

• Can be reversed by reintroducing the wild type protein

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Maize (Corn)- A common biological

model used for genetic studies

-Biello, David. That Burger You’re Eating Is Mostly Corn. Scientific American. Available from :

http://www.scientificamerican.com/article.cfm?id=that-burger-youre-eating-is-mostly-corn

--Corn. University of Illinois Extension: Watch your Garden Grow. Available from:

http://urbanext.illinois.edu/veggies/corn.cfm -McGinnis KM et al., (2006) Genetics, 173:1637-1647. -ZeaMays. Wikipedia. Available from

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RMR2 in Regulation of Gene Expression in Maize

McGinnis KM et al., (2006) Genetics, 173:1637-1647.

RMR1 & RMR2

RMR2

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Aim of the project

To study the role and expression of RMR2 on a

molecular level

– Fluorescent gene tagging

• DNA sequence coding for fluorescent protein

– Citrine yellow

»

Produced in the protein product

Fluorescent

marker

Protein product

(rmr 2)

Tether

Gene fragment 1 Gene fragment 2

Florescent tag

Jackson, David. Sylvester, Anne. Chan, Agnes. Etc. Maize Cell genomics DB (2010). Available from: http://maize.jcvi.org/cellgenomics/

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Experimental outline

• Use Multi site Gateway cloning to make the construct

– Primer design

– Amplification of Rmr2 DNA fragments

– BP reaction

– Sequencing

– LR reaction

• Introduce the construct into Agrobacterium

tumefaciens

• Transform maize at the Plant Transformation Facility

(ISU)

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Gateway Cloning

• Gateway cloning

• PCR

(polymerase

chain reaction)

• Plasmids

• Special

flanking

sequences

User Manual for MultiSite Gateway Pro (2006) available from:

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Designing primers for Gateway cloning

• Determining the insertion position

– The best position to insert the fluorescent protein is right after

the start codon

• Should incorporate regulatory regions

– 3000 bp upstream

– 800 bp downstream

• Primer 3 (http://frodo.wi.mit.edu/)

– p1:

GGGG ACA AGT TTG TAC AAA AAA GCA GGC T

ct agc cac ttg

gct gta ctg tg

– p4:

GGGG AC AAC TTT GTA TAG AAA AGT TGG GTG

cat ggt acc

ggc ggt ctt gg

– p3:

GGGG ACA ACT TTG TAT AAT AAA GTT GAG

ccg gtc ctc cgc

tcc ccg t

– p2:

GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA

ctt gcc ggt

gca gta gag tt

Steve Rozenand Helen J. Skaletsky. Primer3. (2000) available at http://fokker.wi.mit.edu/primer3/

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p2p3

PCR rmr2 fragments

Products are amplified and results were observed on an agarose gel:

p1p4~ 3200bp p2p3~3800bp

Fig 1

_{p1p4 p2p3}

Fig 2

3000

4000

3000

4000

Fluorescent

tag

p1

p4

p2

p3

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Gateway Cloning

User Manual for MultiSite Gateway Pro (2006) available from:

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Gel purification and BP reaction

•Bands were cut out and gel purified.

•1 µL of each product was loaded on a gel

•BP reaction

was conducted to introduce

the DNA fragments into the pDONOR

vector.

•Kanamycin resistance

•The vectors are then introduced into the

kanamycin sensitive bacteria (E. coli)

through transformation.

•Heat shock

Fig 3

4000

3000

P2P3

P1P4

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• Bacteria were plated on kanamycin plates and

were grown at 37

◦

_{C overnight.}

• Colonies are then subjected to colony PCR

– Amplify the DNA sequences in the plasmid

– Plasmid specific primers

Colony PCR

Fig 3

Fig 4

_{Samples 1-12}

p1p4

Samples 13-23 p1p4

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Colony PCR with

Fresh Primers

Fig 5

Fig 6

p2p3 p1p4 Control “+” 3000 4000 5000 p2p3 p1p4 5000 3000 4000

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Restriction Digest with

HaeIII and HindIII

New DNA

Size site1 site2 _{Mass % 1 Start}

213 HaeIII 229 HaeIII 359 HaeIII 376 HaeIII 667 HaeIII 1172 HaeIII 2581 HaeIII 2933 HaeIII 2955 HaeIII 2990 HaeIII 3118 End 505 HaeIII 667 HaeIII 1172 16 352 HaeIII 2581 HaeIII 2933 11 291 HaeIII 376 HaeIII 667 9 130 HaeIII 229 HaeIII 359 4 128 HaeIII 2990 End 3118 4 35 HaeIII 2955 HaeIII 2990 1 22 HaeIII 2933 HaeIII 2955 1 16 HaeIII 213 HaeIII 229 1 17 HaeIII 359 HaeIII 376 1 212 Start 1 HaeIII 213 7 1409 HaeIII 1172 HaeIII 2581 45 New DNA

3471 HindIII 3900 HindIII 3906 End 3470 Start 1 HindIII 3471 89 429 HindIII 3471 HindIII 3900 11 6 HindIII 3900 End 3906 0 New DNA

3471 HindIII 3900 HindIII 3906 End 3470 Start 1 HindIII 3471 89 429 HindIII 3471 HindIII 3900 11 6 HindIII 3900 End 3906 0

Hae III

Hind III

Davis, M. Wayne. APE. Version 2.0.44. 12 from: http://biologylabs.utah. edu/jorgensen/wayne d/ape/

P1P4

P2P3

3000 4000 400 1000

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Work Progress and Conclusion

 All steps prior to the BP reaction were successful

 Bacteria have the plasmid

 Survived on the kanamycin plates

 Indicated that plasmid is present

 Most of the bands are smaller than expected

 Restriction digest confirmed that fragments of Rmr2 were inserted

 Possible reasons:

 Recombination alteration

 Bacteria excreting plasmids out

 Dimers

 Maize sequence repeats

Future Trouble Shooting

• Use a different bacteria vector

• Utilize only PCR reactions

• New primers

• Avoid Maize genome issues: common repeats, GC rich (different

physical structure)

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Acknowledgment

Special thanks to:

Dr. Anne Sylvester

Dr. Jacque Keele

Dr. Anding Luo

Rest of Sylvester’s Lab

EPSCoR

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Work Cited

• Biello, David. That Burger You’re Eating Is Mostly Corn[internet]. Scientific American. [12 November 2008; 29 March 2012]. Available from : http://www.scientificamerican.com/article.cfm?id=that-burger-youre-eating-is-mostly-corn

• Chandler, Vicki L. Paramutation: From Maize to Mice. Cells [internet]. [2007; cited 12 April 2012]. 128: 641-645. Available from:

ftp://ftpmips.gsf.de/plasmar/epigenetics/Cell%20review%20Issue/Paramutation%20-%20From%20Maize%20to%20Mice.pdf

• Commonly Used Promoters [internet]. African Biosafety Network Expertise. [2010; cited 29 March 2012] available from

:http://www.nepadbiosafety.net/for-regulators/resources/subjects/biotechnology/commonly-used-promoters • Corn [internet]. University of Illinois Extension: Watch your Garden Grow. [2012: 10 April 2012]. Available from:

http://urbanext.illinois.edu/veggies/corn.cfm

• Corn and Sorgnum Pictures/corn ears [internet]. Texas A&M. [1 March 2002; cited 29 March 2012]. Available from: http://soilcrop.tamu.edu/photogallery/cornsorghum+/pages/corn%20ears.htm

• Davis, M. Wayne. APE [internet download]. Version 2.0.44. 12 April 2012. Available from: http://biologylabs.utah.edu/jorgensen/wayned/ape/

• Jackson, David. Sylvester, Anne. Chan, Agnes. Etc. Characterizing Sub-cellular Compartments in Maize Using Fluorescent Protein Tagging Lines. Maize Cell genomics DB. [18 December 2010; cited 20 April 2012]. Available from:

http://maize.jcvi.org/cellgenomics/

• McGinnis KM, Springer C, Lin Y, Carey CC, Chandler V (2006) Transcriptionally silenced transgenes in maize are activated by three mutations defective in paramutation. Genetics, 173:1637-47.

• Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th_{. New York: W. H. Freeman; 2000.}

• Steve Rozenand Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers.In: Krawetz S, Misener S (eds)Bioinformatics Methods and Protocols: Methods in Molecular Biology.Humana Press, Totowa, NJ, pp 365-386 Source code available at http://fokker.wi.mit.edu/primer3/

• User Manual for MultiSite Gateway Pro (Invitrogen 2006) Using gateway technology to simultaneously clone multiple DNA fragments.

Available from: http://www.invitrogen.com/vntigateway

Biological Function of RMR2 in Maize: Genetic Study through Fluorescent Tagging of RMR2 Protein