Transcript Differential Accumulation Pattern of Met-rich beta

Differential Accumulation Pattern
of Met-rich b-zein in Medicago
sativa and Medicago truncatula
Serina Padilla
Dr. Champa Sengupta-Gopalan,
Mentor
Department of Plant and
Environmental Science, New Mexico
State University
Medicago sativa (alfalfa) and the
Importance of Methionine
 Important forage legume for livestock feed
 Low levels of methionine
Methionine deficiency in animals limits wool
growth, milk production, and meat production
(Bagga et al, 2004)
 Livestock owners must supplement feed with
methionine
Most of the methionine is consumed by rumen
microorganisms
Increasing Methionine In
M.sativa
Cross-breeding
Cell selection
Genetic engineering approach: Introduction
of genes encoding for high methionine
protein.
Zeins
 Seed storage proteins of corn
 Four classes:
a (19kD and 22kD) has low levels of methionine
b (15kD) 10% methionine
g (16kD and 27kD) has low levels of methionine
d (10kD and 18kD) 22 and 27% methionine
respectively (Bagga et al, 2004)
 b-zein ideal for introduction into M.sativa through
a gene cassette
Nos 3’
b-zein
CsVMV promoter
PA 24 3’
Medicago sativa Transformation
1.
Leaf segments are inoculated
with Agrobacterium engineered
with -β zein gene construct.
Callusing is observed in leaf
segments within 2-3 weeks
2.
Callus is placed on selection
media (25mg/L kanamycin)
3.
Green embryoids are observed in
callus after 6-weeks and they are
regenerated into plantlets
4.
The transgenic M. sativa plants
appear to be morphologically
normal, produce flowers and can
be nodulated
b-zein protein accumulation in
M.sativa
b- zein is a bypass protein (not broken down
by rumen organisms) but methionine levels
are not high enough to be of nutritional value
(Bagga et al, 2004)
Working Hypothesis
 Make a comparison using a model Medicago
species to find possible differences with regards to
the amino acid composition and the rate of
synthesis of Met rich proteins in the two legumes
of the same species.
 An understanding of the basis for any differences
between M.sativa and the model legume,
M.truncatula, will allow us to increase the Metcontaining proteins in M.sativa using a genetic
engineering approach.
Medicago truncatula
Transformation
1.
M. truncatula leaf and root
segments from seedings are
inoculated with Agrobacterium
engineered with β-zein gene
constructs. Callusing is observed
in leaf and root segments within
2-3 weeks
2.
Callus is placed on selection
media (25mg/L kanamycin)
3.
Green embryoids are observed in
callus after 6-weeks and they are
regenerated into plantlets
4.
The transgenic plants appear to
be morphologically normal.
bzein protein in M.truncatula
independent transformants
Pattern of protein accumulation of β-zein in transgenic M. truncatula leaves was perfomed with Western analysis:
EtOH-soluble fractions (equivalent to 25 ug of PBS-soluble fraction) from M. truncatula leaves were subjected to SDSPAGE followed by immunoblot analysis using b-zein antibodies. Lanes: M. truncatula transformants lanes 7 to 14;
Tobacco transformant expressing b-zein
The transformants with the b-zein gene construct
show higher levels of b-zein accumulation.
Comparison of b-zein Protein
Accumulation
Pattern of protein accumulation of β-zein in transgenic M. sativa and M. truncatula leaves was perfomed with Western
analysis:
EtOH-soluble fractions (equivalent to 25 ug of PBS-soluble fraction) from M.sativa and M. truncatula leaves were
subjected to SDS-PAGE followed by immunoblot analysis using b-zein antibodies. Lanes: M. sativa non-transformed
control, M. sativa 7 and 10 b-zein transformants, M. truncatula non-transformed control, M. truncatula 8 and 14 b-zein
transformants
Medicago truncatula shows ~10-20 fold higher levels of
b-zein protein accumulation compared to Medicago
sativa
Comparison of b- Zein Transcript
Levels
Comparison of transcript levels in M. sativa and M. truncatula was performed by Northern analysis: 20 ug of total RNA from M.
sativa and 10 ug of total RNA from M. truncatula was seperated on the same 1.0% agarose formaldehyde gel and subjected to
gel blot analysis using a 690 bp b-zein fragment as a probe. Blots were also hybridized to M. sativa Glutamine Synthase (GS2)
probe to check the levels of GS2 transcript in M. sativa and M. truncatula. The third panal shows ethidium bromide stained
ribosomal bands. Lanes: M. sativa not-transformed control, M. sativa 7 and 10 b-zein transformants, M. truncatula nontransformed control, M. truncatula 8 and 14 b-zein transformants.
The M. truncatula transformants show approximately 20
fold higher levels of b-zein expression compared to M.
sativa expressing the same gene.
Methionine Pathway
Conclusion
Metabolite analysis shows M.sativa has
high levels of S-adenosylmethionine
(SAM)
Down regulation of SAM synthase could
increase b-zein production
Acknowledgements
Dr. Suman Bagga
Matt Barrow
Omar Holguin
References
Bagga, S, Armendaris, A, Klypina, N, Ray, I, Ghoshroy, S, Endress, M, Sutton,D.,
Kemp, J.D., and Sengupta-Gopalan, C.(2004). Genetic engineering ruminal stable
high methionine protein in the foliage of alfalfa. Plant Sci. 166, 273-283.
Questions