Transcript RR - UBC Blogs

The wrinkled-seed character of pea described
by Mendel is caused by a transposon-like
insertion in a gene encoding starch-branching
enzyme.
Bhattacharyya et al, 1990
Storage compounds are located in
the embryo of dicotyledonous seeds
Seed coat
Endosperm
Embryo
Amylose and Amylopectin differ in their
branching
Amylose (Alpha-1,4 glucosidic linkage)
Amylopectin (Alpha-1,4 glucosidic linkage& Alpha-1,6 glucosidic linkage)
2-D Representation of Amylopectin
Reducing end
Non-reducing end
Alpha-1,4 glucosidic linkage
Alpha-1,6 glucosidic linkage
Differences Between RR and rr
rr
RR
round
wrinkled
Less sweet low sucrose
Sweeter high sucrose
More starch
Less starch
Amylopectin/amylose high
low
low
High water content
Lower lipid higher protein
Higher lipid lower protein
Cell size smaller lose less volume
Larger lose more volume
Larger simple starch grains
Smaller compound
More legumin
less
Lower
Higher osmotic pressure
SBEI
No SBEI
Table 1. Composition of Mature RR
and rr Pea seeds differ in many ways
• Ranges refer to studies on a number of genotypes (given in paranthesis)
• a, b and c indicate paired studies with near isogenic lines of RR and rr plants
• % dwt indicates percent dry weight
Figure 1. SBEI Antiserum Cross Reacts with Purified
SBEI and Crude Extracts of Pea Embryos
SDS-PAGE
(a) Purified SBE I
WESTERN
(Antiserum)
WESTERN
Preimmune
serum)
(b) SBE I in crude embryo extracts
Cloning of the SBEI Gene from Pea
bacteriophage λ gt11– a vector for cloning and expressing
in bacteria cDNAs derived from any organism.
Cloning site
Bacterial
promoter
λ
P
λ
+ cDNA
λ
P
cDNA
λ
Cloning of the SBEI Gene from Pea
Pea embryo cDNA
+ λ gt11 =
pea embryo expression library.
--each λ gt11 has a cDNA for a different gene expressed in
the pea embryo.
λ
P
cDNA
λ
Screening the λ gt11 library
Petri plate
top view
Bacteria lawn
+
λ gt11 library
Nitrocellulose
filter
Expose to SBEI antibody and detect.
Cloning of the SBEI Gene from Pea
Following screening of λgt11 Pea library:
--7 λgt11 clones identified.
--all clones contained cDNA sequence from a single gene.
-- with sequence similarity to bacterial glycogen branching
enzyme.
Is it the SBEI gene and if so is SBEI the R gene?
Figure 2. RNA blot Analysis of SBE I
(a) Restriction map of putative SBEI cDNA
(c) RNA from different stages of
development of pea embryo
(b) RNA from RR and rr embryos
Figure 3. SBEI RFLP’s Between Near Isogenic Lines of
Pea RR and rr Can Be Observed
The SBEI Gene and R Gene Cosegregate
Probe = 1.3 kb
EcoR1 cDNA fragment
Figure 5. Analysis of the SBE I Gene from
RR and rr plants
RR gene
rr gene
Figure 6. Sequences of the Terminal Inverted
repeats of the Insertion Element Ips-r
(a) Sequences of 12 bp inverted repeats
and flanking 8 bp direct repeats
(b) Homology of the Ips-r inverted repeats
with those of other elements
Conclusions
• The R gene encodes a starch branching enzyme.
• The r allele results in a wrinkled phenotype because a
transposable element insertion in the SBEI gene
eliminates SBEI activity causing a decrease in
amylopectin.