Transcript Document 7583371

Molecular aspects of early steps in steviol biosynthesis
Amal A.A. Mohamed, Jan M.C. Geuns, Wim Van den Ende & Marc De Ley
Proceedings of the 3rd Stevia symposium, organised by EUSTAS
July 1st and 2nd 2009
History
- In 1999, Richman et al. cloned the first two genes- copalyl diphosphate synthase
(CPS) and kuerene synthase (KS) –in the committed steps leading to the synthesis of
gibberellins and steviol.
- They declared presence of one copy of CPS and two copies of KS
- They profiled their expression patterns in both old and young leaves using northern
blotting and claimed that their expressions are high in old leaves and owed that to the
contribution of these two genes in steviol glycosides biosynthesis which present in
quantatities of 107 in comparison to gibbrillins.
- They couldn’t interpret the mechanism for regulation between synthesis of
gibberellins and steviol gylcosides in the light of their outputs.
Aims
- Profiling the expression patterns of these two genes -using real time quantitative
polymerase chain reaction (RT-q-PCR) technique- in Stevia rebaudiana plants grown at
different light conditions.
- Comparing their expressions between old and young leaves.
- Estimation of the accumulation patterns of the steviol glycosides in these plants.
1.1. Methods for profiling gene expression
1.
Introduction
2. Materials and Methods
Northern blotting
Nuclease protection assays
RT-q-PCR
3. Results and Discussion
4. Conclusion
5- Future Perspectives
-is the most sensitive technique
-to quantify mRNA levels from much smaller samples.
-sensitive enough to enable quantitation of RNA from a single cell.
1.2. Biosynthetic pathway to steviol and steviol glycosides
1.
Introduction
Bank accession numbers:
CPS: AF034545;
KS1-1 & KS22-1:
AF097310 &
AF097311,respectively
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
1.3. Designing the primers
1.
Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
-For studying the expression by RT-q- PCR, specific characters for
primers should be achieved:
1.4. Primers for KS1.1 and KS22-1 in an untranslated
region
1.
Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
KS1-1
atttcttttgctctgattgaacacacgattatc
tgtatttatgaggattgtagcatattgtttgat
tttttatgattattttacacttattctctgtca
tctccgagtctatttggtctgacaagcaataca
atatcctcttagattcactctacataggattta
ggatgcattgttattatcctaaaatcatcttca
ttttgatccattgcactgcctgctccgtaagct
tcttatgattcggataattccggtagagaataa
agttcggagtatgcatcattgaagttaatattc
tggattgatgttcgttaggaattggtacagtag
aaaattagctagtttgcttgatttgacctagaa
tatagcttcagaaatttgttaatctgtgagcaa
tgattcagatgttgataacttttggggcaactt
catgttaaaaaacttatatattaaaatgttttc
aagttggtgttgcagtaaatcagcaaactaaac
atca
Translated region
atgaatctttcactatgcattgcgtcccctttg
ttaaccaaatcaagtcgacccacggctctgtca
gctattcatacagcatcaacttcacatggtgga
caaactaatcccactaatctgatcattgataca
accaaagaacggatccaaaaactgtttaaaaat
gtagaaatttctgtttcttcatatgacacagca
tgggtagccatggtcccttctccaaactcaccc
aaatcgccttgtttccctgagtgtctcaattgg
ttaattaataatcagcttaatgatggttcatgg
ggtcttgttaatcacactcataatcataatcac
ccgttgcttaaagattctctatcttcaacatta
gcatgtattgttgcattaaaaagatggaatgtt
ggggaagatcaaataaataaaggtctaagtttt
attgagtcaaatcttgcttcagcaactgacaaa
agtcaaccatctcccattggttttgatatcata
tttcctggtttgcttgagtatgcgaaaaacttg
gacataaacctcctttcaaaacaaacagatttt
agtttgatgctacataagagggaattggagcaa
aaaagatgccattcaaatgagattgatggatac
ttggcgtatatctctgaaggactcggtaattta
tatgattggaatatggtgaagaaatatcagatg
aaaaatggttctgttttcaactcaccatcagca
acagcagctgctttcattaatcatcaaaatccc
ggttgtcttaattatttaaattcacttttggac
aagtttggtaatgcagtcccaacagtttatcct
cttgatttatatatccggctttctatggttgac
acaattgaaagattaggaatttcacaccatttc
agagtggaaattaaaaatgttttagatgaaaca
tacagatgttgggtggaacgagatgagcaaata
ttcatggatgttgtaacatgtgctttagccttt
cggttattaaggatccacgggtataaagtctcc
ccagatcaattggctgaaattactaatgaatta
gctttcaaagacgaatacgcagctcttgaaaca
tatcatgcatcacagatattataccaagaggat
ttatcttctggaaaacaaatcttgaagtcagct
gatttcctcaaagggatattatccactgattca
aacaggctttctaaattaattcacaaagaggtg
Ks22-1
cccactcatcctttatcaaccaataccatcgttc
tgccaccggaagactgatacgcgacggacctgac
gacgcctttaatctctgttggaccacctaacaaa
attccgaccatcagatctactccggtggacagtt
tcatttgcaatttagtaaatcagcaaactaaaca
tca
atgaatctttcactatgcatcgcgtcccctttg
ttaaccaaatcaaatcgacccgcggctctgtca
gctattcatacagcatcaacttcacatggtgga
caaactaatcccactaatctgatcattgataca
accaaagaacggatccaaaaacagtttaaaaat
gtagaaatttctgtttcttcatatgacacagca
tgggtagccatggtcccttctccaaactcaccc
aaatcgccttgtttccctgagtgtctcaattgg
ttaattaataatcagcttaatgatggttcatgg
ggtcttgttaatcacactcataatcataatcac
ccgttgcttaaagattctctatcttcaacatta
gcatgtattgttgcattaaaaagatggaatgtt
ggggaagatcaaataaataaaggtctaagtttt
attgagtcaaatcttgcttcagctactgaaaaa
agtcaaccatctcccattggttttgacatcata
tttcctggtttgcttgagtatgcgaaaaacttg
gacataaacctcctttcaaaacaaacagatttt
agtttgatgctacataagagggaattggagcaa
aaaagatgccattcaaatgagatggatggatac
ttggcgtatatctctgaaggactcggtaattta
tatgattggaatatggtgaagaaatatcagatg
aaaaatggttctgttttcaactcaccatcagca
acagctgctgctttcattaatcatcaaaatcct
ggttgtcttaattatttaaattcacttttggac
aagtttggtaatgcagtcccaacagtttatcct
catgatttatttatccgactttctatggttgac
acaattgaaagattaggaatttcacaccatttc
agagtggaaattaaaaatgttttagatgaaaca
tacagatgttgggtggaacgagatgagcaaata
ttcatggatgttgtaacatgtgctttagccttt
cggttattaaggatcaatgggtatgaagtttcc
ccagatccattggctgaaattactaatgaatta
gctttgaaagacgaatatgcagctcttgaaaca
tatcatgcgtcacatatattataccaagaggat
ttatcttctggaaaacaaatcttgaagtcagct
gatttcctcaaagagataatatccactgattca
aacaggctttctaaattaattcacaaagaggtg
1.5. Choosing the house keeping gene
1.
Introduction
The most internal controls used in RT-q-PCR
are
2. Materials and Methods
3. Results and Discussion
4. Conclusion
18S rRNA
5- Future Perspectives
is being recommended as the best internal control because it
shows less variance in expression than B-actin and GAPDH.
1.6. Choosing the detector
1.
Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
- SYBR green provides the simplest and most economical format for detecting
and quantitating PCR.
1.
Introduction
2. Materials and Methods
Adventages
of SYBER Green
Disdventages
of SYBER Green
- Inexpensive
- Easy to use
Inspecifically bound to any
double strand in the reaction
including primer dimer.
- Sensitive
3. Results and Discussion
But
4. Conclusion
5- Future Perspectives
- For single PCR product reactions with well designed primers, SYBR Green can
work extremely well, with spurious nonspecifiec background only showing up in
very late cycles.
1.7. Three outputs for succesful Q-PCR reaction:
1.
Introduction
1- Dissociation curve should has one
peak at the same melting temperature
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
2- Amplification plot the different
dilutions cross the threshold line at
equal distance of cycle number
3- The strandard curve for the relation
between log of cencentration of
different dilution folds and Ct value
should has a value of the slope not less
than -3.2 and the r2>0.99
2.1. Methodology
1.
Introduction
2. Materials and Methods
RNA
extraction
RNA
RT
cDNA
House
keeping
gene
12/12 light/dark
16 light/8 dark
Target
gene 1
CPS
Target
gene 2
(KS1)
3. Results and Discussion
4. Conclusion
5- Future Perspectives
RT-q-PCR
instrumentation
Data analysis
3.1. Comparison of transcript levels of SrCPSent in both
young and old leaves
1.
Introduction
1
3
2
2. Materials and Methods
5
3. Results and Discussion
6
4
4. Conclusion
5- Future Perspectives
1
Three weeks young leaves
2
Four weeks young leaves 3
4
Three weeks old leaves
5
Four weeks old leaves
6
Two weeks young leaves
Two weeks young leaves
3.2. Dissociation curve showing melting temperature for CPS
1.
Introduction
2. Materials and Methods
It shows one
melting temperature
for the standard
series
But!
3. Results and Discussion
4. Conclusion
5- Future Perspectives
For some samples
( specifically in old
leaves), a different meting
temperature was
noticed.This may suggest
a presence of another
copy of CPS according to
Totté et al. (2003)
3.3. The expression patterns of ent- CPS:
1.
Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
Young Leaves
The expression
declined after the
first week in both
old and young
leaves
3.4. The expression patterns of ent-KS:
1.
Introduction
young
leaves
old
leaves
There is no big
difference in the
KS expression
beween young
and old leaves
25.00
25.00
3. Results and Discussion
4. Conclusion
5- Future Perspectives
KS1/Actin
KS1
/Actin
2. Materials and Methods
20.00
20.00
15.00
15.00
10.00
10.00
5.00
5.00
0.00
0.00
1
1
2
3
3
4
Time (Weeks)
Time (w eeks)
4
2
b16/8full b12/12full b12/12 shade b16/8shade
y16/8full 1212full y12/12shade y16/8shade
d
3.5. Accumulation of different steviol glycosides in comparison to CPS
expression patterns in young leaves: (a)16/8 full light, (b) 16/8 shady,
( c )12/12 full light
(b)
(a)
Introduction
2. Materials and Methods
0.30
2
0.20
1
0.10
0
ST.glycoside %
0.40
cps/actin
ST.glycoside %
3
3
0.00
1
2
3
0.03
0.02
2
0.02
0.01
1
0.01
4
0
Tim e (w eeks)
0.00
1
2
3
Tim e (w eeks)
(c)
4. Conclusion
0.80
0.60
2
0.40
1
0.20
0
0.00
1
rebaudioside A
5- Future Perspectives
cps/actin
3
ST.glycoside %
3. Results and Discussion
2
3
Tim e (w eeeks)
stevioside
4
rebaudioside C
cps
4
cps/actin
1.
1.
Introduction
The expression of SrCPS ent was very small in old leaves in comparison to
young leaves. The presence of one copy of the gene to trigger the biosynthesis
of steviol and gibberellic acid at the same time with an unknown mechanism of
regulation (Richman et al., 1999) is ambiguous.
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
The experiments showed that there was no big variance in SrKS ent expression
between old leaves and young leaves and this confirmed the tight control of
CPS activity over KS in the pathway (West et al., 1982; Yamaguchi et al., 1996;
Hedden, 1999).
1.
Introduction
To design the primers
for RT-q-PCR
profiling of KO, KAO
& KAH
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5- Future Perspectives
References
Hedden, P. (1999) Recent advances in gibberellin biosynthesis. Journal of Experimental Biology
50(334), 553-563.
Richman, A. S.; Gijzen, M.; Starrat, A. N.; Yang, Z. and Brandle, J. E.(1999). Plant J. 19, 411-421.
Totté, N.; Van den Ende, W.; Van Damme, E. J. M.; Compernolle, F.; Baboeuf, I. and Geuns, J.
M. C. (2003) Can. J. Bot. 81, 517-522.
West, C.A.; Shen-Miller, J. and Railton, I. D. (1982) Regulation of Kaurene synthase. In:
Wareing PF, ed. Plant growth substances 1982. London: Acadmic Press,81-90.
Yamaguchi, S.; Saito, T.; Abe, H.; Yamane, H.; Murofushi, N. and Kamiya,Y. (1996).The Plant Journ
10, 203-213.
Mr. Tom Struyf
Mrs. Hilde Verlinden
Mrs. Sofie Van Soest
Acknowledgements
Mrs. Pegah Maghdooni Bagheri
Mr. Stijn Ceunen
Thanks for your attention!