1. 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

2. Storage compounds are located in the embryo of dicotyledonous seeds
Seed coat
Endosperm
Embryo

3. Amylose and Amylopectin differ in their branching
Amylose (Alpha-1,4 glucosidic linkage)
Amylopectin (Alpha-1,4 glucosidic linkage& Alpha-1,6 glucosidic linkage)

4. 2-D Representation of Amylopectin
Reducing end
Non-reducing end
Alpha-1,4 glucosidic linkage
Alpha-1,6 glucosidic linkage
osmotic pressure
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. Glycogen is a more highly branched molecue than amylopectin-otherwise they are similar molecules.

5. Differences Between RR and rr
round
wrinkled

6. Differences Between RR and rr
round
wrinkled
Less sweet low sucrose
Sweeter high sucrose
More starch
Less starch
Amylopectin/amylose high
Amylopectin/amylose low
Lower water content
Higher water content
Lower lipid higher protein
Higher lipid lower protein
Cell size smaller lose less volume
Cell size larger lose more volume
Larger simple starch grains
Smaller compound starch grains
More legumin
Less legumin
Lower osmotic pressure
Higher osmotic pressure
SBEI
No SBEI

7. 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

8. Isogenic lines (identical genetic backgrounds except for R vs r)
(AA bb) RR x rr (aa BB) Back Cross 1 (BC1) Rr x RR. BC2 RR and Rr self self RR disgard 25% rr x RR BC Rr x RR Continue Back crossing for at least 7 generations. This will result in the genotypes of the two lines (RR vs rr) being identical except at the R locus

9. Figure 1. SBEI Antiserum Cross Reacts with Purified SBEI and Crude Extracts of Pea Embryos
WESTERN
Preimmune
serum)
WESTERN
(Antiserum)
SDS-PAGE
(a) Purified SBE I
(b) SBE I in crude embryo extracts

10. ’R’ – SBEI Pick the best answer! ***iClicker!
Based on the data in the previous slide what can be concluded about the relationship between R and SBEI?
The R gene encodes the SBEI protein.
B. The R gene function is required for the SBEI protein.
The R gene and SBEI are separate loci.
D. The SBEI gene influences the R gene.
E. All of the above.

11. 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
A cDNA from one gene λ

12. cDNA synthesis

13. 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

14. Screening the λ gt11 library
Petri plate
top view
Bacteria lawn +
λ gt11 library
Nitrocellulose
filter
Expose to SBEI antibody and detect.

15. 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?

16. List experiments that were done that establish a connection between R and SBEI.-

17. Figure 2. RNA blot Analysis of SBE I
(a) Restriction map of putative SBEI cDNA
(b) RNA from RR and rr embryos
(c) RNA from different stages of
development of pea embryo

18. Figure 3. SBEI RFLP’s Between Near Isogenic Lines of Pea RR and rr Can Be Observed
Probe used was the entire 2.7 cDNA

19. The SBEI Gene and R Gene Cosegregate
Probe = 1.3 kb
EcoR1 cDNA fragment

20. Figure 5. Analysis of the SBE I Gene from RR and rr plants
RR gene
rr gene

21. Figure 6. Sequences of the Terminal Inverted repeats of the Insertion Element Ips-r
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

22. 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.