Developing a binary vector for sweetpotato intragenesis

1. Developing a binary vector for sweetpotato intragenesis Jose Carlos Tovar

2. Intragenesis • Is crop improvement by genetic engineering, using only the crop’s own genome; • Implies the use of DNA sequences which exist in the crop’s species.

3. Intragenesis • We need a binary vector with a T-DNA made from sweetpotato sequences. • Sequences are put together so that they form the required element.

4. Elements needed • A multiple cloning site; • A selectable marker; • P-DNA borders.

5. 1. Multiple cloning site • Sweetpotato sequences with 2 or 3 “common use” restriction sites were pasted together.

6. 2. A selectable marker • No selectable marker was found in the sweetpotato genome; • Cre-lox system; • lox-like sites were built.

7. lox-like sites • Lox 71: taccgTTCGTATA ATGTATGC TATACGAAGTTAT atccgTTCGTATA AATTAGCA TATACGAAGTTAT • Lox 66: ATAACTTCGTATA ATGTATGC TATACGAAcggta ATAACTTCGTATA AATTAGCATATACGAAcggat • After excision: atccgTTCGTATAAATTAGCATATACGAAcggat

8. 3. Border-like sequences • 25 bp conserved sequences were used for each border; • A proposal from the NZ group was used.

9. Intragenic construct

10. Binary vector

11. Testing of the elements • Every sweetpotato fragment was sequenced to verify integrity; • lox-like sites were functional: intragenic control

12. Testing of the elements • Borders were tested by assaying for GFP expression in N. benthamiana. • No GFP activity was found; • Borders do not work. intragenic control

13. Why don’t they work? • Border sequences used are the same as in functional vectors, such as the pBIN or pCAMBIA series; • Sequences adjacent to the 25 bp repeat may be important; • What are these sequences?