Chloroplast Genome Transformation Revital S.G., Edo M., Stefan L., - - PowerPoint PPT Presentation

Genetic Engineering of H. pluvialis by Nuclear and Chloroplast Genome Transformation

Revital S.G., Edo M., Stefan L., Aliza Z. and Sammy B.

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation

Potential

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 2

 Haematococcus pluvialis (H. pluvialis) accumulate up to 4%

astaxanthin on a dry weight basis (Boussiba, 2000).

 Limitations:

 Low growth rate  Environmental stresses

These limitations might be mitigated, by genetic engineering

Goals and Achievements

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 3

 Nuclear:

 pBS-PDS S/L  pBS-PDS S/L-ble 5’  pBS-PDS S/L-ble 3’  PDS S/L  PDS S/L-ble 5’

 Chloroplast:

 pUC-atpX-aadA-16S-23S

We have developed nuclear and chloroplast transformation systems for genetic engineering of H. pluvialis.

Circular Linear

Nuclear vectors introduced

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 4

Transformed colonies

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 5

Control pBS-PDS S pPlat-PDS

Transformed cultures

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 6

Optimal conditions HL

• N

PDS gene is present in several pBS-PDS /linear PDS- transformed colonies

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 7

1-3- pBS-PDS-S 14-pBS-PDS S 4-6- pBS-PDS-L 15-pBS-PDS L 7-9- PDS-S linear 16,17 - pBS-PDS-S/L+13 10-12- PDS-L linear 18-no DNA 13- H. pluvialis gDNA 19-spontaneous resistant colony M– marker colonies

Mutation site as sequenced from transformed colonies

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 8

Polymorphism in PDS introns indicate successful transformation

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 9

pBS-PDS as a versatile tool for nuclear transformation

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 10

 PDS* cassette serves as an efficient selection marker for

nuclear genome transformation.

 linear and circular DNA are inserted efficiently.  Short and long promoter versions are equally effective.

Insertion of additional gene

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 11 (psaD-ble cassette was driven from the pGenD-ble of Fischer and Rochaix, 2001)

ble gene is present in transformed colonies

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 12

1-5- pBS-PDS-L-ble 3’ 18, 19- pBS-PDS-S-ble 5’/3’, respectively 6-8- pBS-PDS-S-ble 5’ 20, 21- pBS-PDS-L- ble 5’/3’, respectively 9-11- pBS-PDS-S-ble 3’ 22-H. pluvialis gDNA 12, 13- PDS-S-ble 5’ 23-no DNA 14-17- PDS-L-ble 5 M– marker colonies

Insertion of ble via pBS-PDS-ble

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 13

 linear PDS-ble is sufficient for transformation.  Ble cassette was incorporated efficiently both at the 5’

and 3’ end of the PDS cassette in pBS-PDS.

 PDS-ble-transformed colonies grew slower than PDS

transformed colonies. This may be due to the heterologous nature of ble or to an inhibitory effect of ble itself.

 pBS-PDS is suitable for the insertion of two or more

transgenes.

Vector for Chloroplast Transformation

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 14

pUC-atpX-aadA was kindly provided by (Goldschmidt-Clermont et al., 1991) based on the method presented in (Gutiérrez et al., 2012).

aadA gene is present in transformed colonies

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 15

1-4- pUC-atpX-aadA-16S-23S-transformed colonies 5– H. pluvialis gDNA 6- pUC-atpX-aadA-16S-23S 7- H. pluvialis gDNA+pUC-atpX-aadA-16S-23S 8– no DNA M– marker

pUC-atpX-aadA-16S-23S is suitable for chloroplast transformation

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16

 The aadA cassette serves as efficient selection marker for

chloroplast transformation in H. pluvialis.

 Insertion into other locations of the chloroplast genome

should be tested.

 Other marker genes for chloroplast transformation

should be tested.

Transformation results

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 17

Vector introduced Bombarded cells (*106) Resistant colonies* Frequency of resistant cells (*10-6) Average (per plate) +Standard Error

No DNA 3 1 0.33 0.5±0.25 pBS-PDS S 6 94 15.7 23.5±3.9 pBS-PDS L 6 111 18.5 27.75±2.8 PDS S linear 6 61 10.2 15.25±2.9 PDS L linear 6 88 14.7 22±5.5 pBS-PDS S-ble 3’ 6 29 4.83 7.25±2.4 pBS-PDS L-ble 3’ 6 58 9.67 14.5±1.25 pBS-PDS S-ble 5’ 6 39 6.5 9.75±1.6 pBS-PDS L-ble 5’ 6 5 0.83 1.25±0.7 PDS S-ble 5’ 6 18 3 4.5±0.6 PDS L-ble 5’ 6 25 4.17 6.25±1.6 No DNA 3 1 0.33 0.5±0.25 pUC-atpX-aadA- 16S-23S 6 20 3.3 5±3

Conclusions

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 18

 All transformation experiments with the various vectors were

successful, both for the nuclear– and for the chloroplast genome.

 Transformation frequencies of about 2*10-5 were achieved,

compared to 2*10-6 reported before.

 Transformation frequency of pBS-PDS S/L and linear PDS are similar.  Transformation frequency of vector+ ble cassette was lower than

vector alone.

 Transformation frequency of pUC-atpX-aadA-16S-23S was lower

than of pBS-PDS S/L or linear PDS.

Concluding Remarks

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 19

 Genetic transformation both to the nuclear- and chloroplast genome of

• H. pluvialis is now established and can be routinely applied.

 Draft genome sequence allowed us to identify and sub-clone essential

genes for basic understanding and to improve the industrial performance of the high-value commercial alga by genetic engineering.

 MBL and GIAVAP have established H. pluvialis as a model organism for

experimental biology and industrial application.

 Additional marker genes need to be tested both for the nuclear

genome and for the chloroplast.

16/09/2013

Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 20

Thank you!!!!