Round/ Wrinkled

Question 1

There is less starch in ____peas. But amylose contains are higher then RR why?

Answer 1

rr-wrinkled
Starch contains amylose and amylopectin so rr peas must be lacking in amylopectin. This can be explained by starch-branching enzymes, SBEs

Question 2

Difference between amylose and amylopectin?

Answer 2

starch is made of glucose polymers linked by glycosidic binds. Amylose and amylopectin are polymers. Amylopectin is branched

Question 3

What explains the lack of amylopectin and a build up of sucrose and amylose?

Answer 3

SBEs

Question 4

——is used up in the generation of amylopectin

Answer 4

amylose

Question 5

Smith(1988) found SBE activity in ——-fractions from round peas and —-fraction from wrinkled.

Answer 5

two fractions
one fraction

There seems to be two SBEs in round peas

Question 6

Round peas have ____SBEI activity peaks and wrinkled has ____

Answer 6

two
one

Question 7

When the round activity peaks are run separately the ___had two bans with molecular weights of ____, and the second peak has on band(100). The ____ were missing from the wrinkled peas.

Answer 7

first peak
108 and 114
first peak and the 108 and 114 bands

Question 8

Isoform

Answer 8

any of two or more functionally similar proteins that have a similar but not identical amino acid sequence and are either encoded by different genes or by RNA transcripts from the same gene which have had different exon removed.

Question 9

Smiths conclusion

Answer 9

A missing SBE isoform explains the low SBE activity levels during early stages in development in wrinkled peas….round peas get a head start on starch/amylopectin synthesis and the wrinkled can’t catch up because they only have one of the SBE isoforms

Question 10

Western Blot

Answer 10

protein is run on a gel and “blotted”. You use a antibody to find out where your protein of interest is in the gel.

Question 11

Bhattacharyya(1990) used ____to label the SBE1 in____peas.
The next stain was using a _____showing that the antibody reacts with an____in RR peas.

Answer 11

Coomassie Brilliant Blue R (CBBR)
Round RR peas

Western Blot
SBE1 found only in

Question 12

How do you make the expression library?

Answer 12

All mRNA from the pea is isolated and turned into cDNA via rtPCR. Next each is cloned into an expression vector. They do this by using phage or bacterial plasmids.

The expression factors express the cDNA, so they make the protein it encodes. Then use the SBE1 antibody to find the clone that has the gene for your particular protein. Finally you sequence the clone.

Question 13

By using the cDNA expression library you get?

Answer 13

you get a cDNA sequence for the functional gene/SBE1

You have this b/c we have identified the expression vector with the cDNA that made a protein that is recognized by your antibody.

Next we use the cDNA to survey mRNA that was isolated from Round and wrinkled peas

Question 14

Norther blot?

Answer 14

transfer RNA from a electrophoresis gel to a membrane(blotting) followed by a hybridization reaction that involved a labeled probe(our case is single-stranded cDNA)

Question 15

Southern blot

Answer 15

transfer of DNA from the electrophoresis gel to a membrane. This DNA was isolated from plant tissue, digested with a restriction enzyme, and then run on a gel. Lastly, it is studied with a labeled probe.

Question 16

How do do southern blot in pea RR and rr

Answer 16

The DNA was isolated from pea plants. Doesn’t matter what tissue because all cells have all the DNA. Then the DNA was digested with three different restriction enzymes, ECOR1, EcoRV, and HIND111

Question 17

What was found on the southern blot in RR and rr peas. Involving EcoR1

Answer 17

It is the 3’ end that produces the variation at 3.3kb and 4.1kb(in rr). Thus, the second half of the genomic DNA SBE sequence is longer in rr plants than it is in RR plants, just like the cDNA was in Northern Blot.

Question 18

What happened to make the 3’ end of the SBE1 gene in wrinkled peas longer? WHat did they do?

Answer 18

Clone the 3’ fragment from RR and rr. Use the restriction enzymes again.(Xbal and Hindlll)

One fragment of the fragment was longer in the rr clone. Between the two Hindlll cut sites it had matches all over the genome.

Using the fragments between the Hindlll cut site from RR plant

Question 19

There is a gene in the genomes of wrinkled and round peas called___

Answer 19

SBE1

Question 20

Peas with one copy of this SBE1 gene(insertions allele) can make a enough SBE1 to produce _____, but when both alleles have the insertion, there’s _______, the pees are sweet, and the sweet coat is wrinkled.

Answer 20

amylose and amylopectin starch
much less amylopectin in the starch

Question 21

Turns out that this insertion does not ____the gene from being transcribed but it does appear to stop translation

Answer 21

completely stop