Answer Key Reverse Genetics Problem

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 300- UTR3 and 1000-Promoter ones are the least favorable since they usually do not affect the target gene.