Transcript Biological Function of RMR2 in Maize: Genetic Study through

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:
Dr. Anne Sylvester
Outline
• Introduction
• Experimental outline and procedures
• Experimental results
• Conclusion
Central Dogma of Biology
• DNA
(deoxyribonucleic
acid)- genetic code
• TranscriptionDNAmRNA
• TranslationmRNA protein
This is followed by Mendelian
inheritance
New Discoveries in genetics
• Silencing
B-I*
• Epigenetic regulation- heritable
changes in gene function without a
change in DNA sequence
Genes can be regulated by:
•
•
•
•
DNA Methylation
Histone acetylation/deacetylation &
methylation
TGS and PTGS (Transcriptional
Genomic Silencing and Post
Transcriptional Genomic Silencing,
Plants)
dsRNA (RNAi)
B’
X
B-I*
B-I*/B-I* B-I*/B’
B’
B-I*/B’
B’/B’
• Paramutation
– An interaction between alleles of
genes that leads to heritable changes
in gene expression
-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.
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
McGinnis KM et al., (2006) Genetics, 173:1637-1647.
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=thatburger-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
:http://en.wikipedia.org/wiki/File:ZeaMays.jpg
RMR2 in Regulation of Gene Expression in Maize
RMR2
RMR1 & RMR2
McGinnis KM et al., (2006) Genetics, 173:1637-1647.
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
Florescent tag
Gene fragment 1
Gene fragment 2
Tether
Protein product
(rmr 2)
Jackson, David. Sylvester, Anne. Chan, Agnes. Etc. Maize Cell
genomics DB (2010). Available from: http://maize.jcvi.org/cellgenomics/
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)
– Stable transgenic plant
Gateway Cloning
• Gateway cloning
• PCR
(polymerase
chain reaction)
• Plasmids
• Special
flanking
sequences
User Manual for MultiSite Gateway Pro (2006) available from:
http://www.invitrogen.com/vntigateway
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
Promoter
tag
Terminator
• Primer 3 (http://frodo.wi.mit.edu/)
– p1: GGGG ACA AGT TTG TAC AAA AAA GCA GGC Tct 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/
PCR rmr2 fragments
Products are amplified and results were observed on an agarose gel:
p1p4~ 3200bp p2p3~3800bp
Fig 1
p1p4
Fig 2
p2p3
p2p3
4000
3000
4000
3000
p1
p4
Fluorescent
tag
p2
p3
Gateway Cloning
User Manual for MultiSite Gateway Pro (2006) available from:
http://www.invitrogen.com/vntigateway
Gel purification and BP reaction
Fig 3
P2P3
P1P4
•Bands were cut out and gel purified.
4000
3000
•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
Colony PCR
• 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
Fig 3
Fig 4
Samples 1-12
p1p4
Control “-”
Samples 13-23
p1p4
Colony PCR with
Fresh Primers
Fig 5
Fig 6
p2p3
p1p4
5000
4000
3000
5000
4000
3000
Control “+”
p2p3
p1p4
Restriction Digest with
HaeIII and HindIII
site2
HaeIII
HaeIII
HaeIII
HaeIII
HaeIII
HaeIII
End
HaeIII
HaeIII
HaeIII
HaeIII
2581
1172
2933
667
213
359
3118
2990
2955
376
229
Mass %
45
16
11
9
7
4
4
1
1
1
1
New DNANew DNA
Size
site1
Size
site1
1 Start
Start
HaeIII
1172
2131409
229
HaeIII 3470
359505
HindIII
376
HaeIII 429
HaeIII
667
HindIII
HaeIII
2581
667352
HaeIII 6
291
HaeIII
376
212
1
1172
HaeIII Start
130
HaeIII
229
128
HaeIII
2990
35
HaeIII
2955
22
HaeIII
2933
17
HaeIII
359
16HaeIII HaeIII
213
2581
site2
1
HaeIII
3471
HaeIII
3900
HaeIII
HaeIII
HaeIII
HaeIII
End
HaeIII
HaeIII
HaeIII
HaeIII
Hae III
2933 HaeIII
2955
2990
3118 End
site2
HindIII
2581
HindIII
1172
End
2933
667
213
359
3118
2990
2955
376
229
New DNA
Size
site1
% 1 Start
Mass % Mass
1 Start
Start
3471
89 HaeIII 3470
45
213
229
HindIII
359
3900
11 HaeIII 429
16
376
HindIII
3906
0 HaeIII 6
11
667
9
7
1172 HaeIII
4
4
1
1
1
1
2581 HaeIII
3471 HindIII
2933 HaeIII
2955
2990
3900 End
HindIII
3906
3118 End
1
3471
3900
site2
HindIII
HindIII
End
Hind III
3471
3900
3906
Mass % 1 Start
89
11
0
3471 HindIII
3900 End
HindIII
3906
4000
3000
1000
400
P2P3
P1P4
Davis, M. Wayne.
APE. Version 2.0.44.
12 from:
http://biologylabs.utah.
edu/jorgensen/wayne
d/ape/
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)
Acknowledgment
Special thanks to:
Dr. Anne Sylvester
Dr. Jacque Keele
Dr. Anding Luo
Rest of Sylvester’s Lab
EPSCoR
NSF
Questions?
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
ZeaMays [Internet]. Wikipedia. [2012; cited 29 March 2012]. Available from :http://en.wikipedia.org/wiki/File:ZeaMays.jpg