1 / 13

Developing a binary vector for sweetpotato intragenesis

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

terena
Download Presentation

Developing a binary vector for sweetpotato intragenesis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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?

More Related