Answer key
Reverse genetics problem
Question 1:
None of LB, RB, Intron, Term, and selectable marker is dispensable, because--- LB and especially RB are required for T-DNA insertion by Agrobacterium
- Intron: when co-transcribed will form a loop-like structure and later be spiced out to
form double-stranded RNA to inducing silencing of the target gene
- Term: required for termination of transcription
- Selectable marker: required for selection of transgenic progeny
A possible plan for making the construct:
1. Determine which restriction enzymes to use for cloning. Retrieve SNI1 cDNA
sequence from NCBI PubMed and run it through a Restriction Site program, paying
attention to the four enzyme sites on the two multi-cloning sites. SNI1 cDNA contains
none of the four restriction sites, thus all four enzymes can be used for cloning.
2. Design primer sets for PCR:
For cloning between 35S and intron: Fragment A can be generated by PCR with the
following two primers:
Forward primer: BamHI cut site sequence + SNI1 cDNA starting sequence
Reverse primer: SNI1 cDNA end sequence + XhoI cut site sequence
For cloning between intron and term: Fragment B can be generated by PCR with the
following two primers:
Forward primer: KpnI cut site sequence + SNI1 cDNA end sequence
Reverse primer: SNI1 cDNA start sequence + EcoRV cut site sequence
3. Set up PCR reactions to amplify Fragment A and B using cDNA made from wild-type
Arabidopsis as template. The fragments will be purified after PCR to prepare for later
reactions.
4. Digest Fragment A and vector separately with BamHI and XhoI, run them on Agarose
gel, and retrieve bands from the gel. Ligate the two fragments, transform E.coli, and
select for clones with insert. This becomes the vector with A cloned.
5. Digest Fragment B and vector with A separately with KpnI and EcoRV, run them on
Agarose gel, and retrieve bands from the gel. Ligate the two fragments, transform E.coli,
and select for correct clones. This becomes the vector with both A and B cloned, which is
ready to be put into Agrobacterium for plant transformation.
Question 2:
When you enter At4g18470 on the SALK website provided, you will find a map. When
you click on At4g18470, a long list of items is displayed. The T-DNA lines are the ones
with Type indicating T-DNA or FST (there are also cDNA or EST clones included on the
list). SALK, SAIL, SK, FLAG indicate different producers of the T-DNA lines from
different countries (refer to lecture notes on T-DNA). All the T-DNA lines that are
available around the globe around SNI1 locus are listed below:
1. HITS SALK_126547.19.55.x [
TYPE SALK T-DNA
LOCN 1000-Promotor
2. HITS SAIL_835_D09
TYPE SAIL FST
LOCN 300-UTR5
3. HITS SK28503
TYPE SK FST
LOCN 300-UTR5
4. HITS SAIL_298_H07
TYPE SAIL FST
LOCN Exon
5. HITS SALK_018281.44.15.x
TYPE SALK T-DNA
LOCN Exon
6. HITS FLAG_186G12
TYPE FLAG FST
LOCN Intron
7. HITS SAIL_34_D11
TYPE SAIL FST
LOCN Intron
8. HITS GABI_869C12
TYPE GABI-Kat FST
LOCN 300-UTR3
The most preferable ones are the ones with insertions in exons (SAIL_298_H07 and
SALK_018281), since the T-DNAs will most likely disrupt full-length protein
production. The ones with T-DNAs in introns or 300-UTR5 are the next favorable ones,
these T-DNAs are likely to abolish or reduce the level of gene transcription. The 300UTR3 and 1000-Promoter ones are the least favorable since they usually do not affect the
target gene.