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

Potential  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 2 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Goals and Achievements We have developed nuclear and chloroplast transformation systems for genetic engineering of H. pluvialis .  Nuclear:  pBS-PDS S/L Circular  pBS-PDS S/L-ble 5 ’  pBS-PDS S/L-ble 3 ’  PDS S/L Linear  PDS S/L-ble 5 ’  Chloroplast:  pUC- atpX-aadA -16S-23S 3 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Nuclear vectors introduced 4 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Transformed colonies Control pBS-PDS S pPlat-PDS 5 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Transformed cultures Optimal conditions HL -N 6 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

PDS gene is present in several pBS- PDS /linear PDS- transformed colonies 1-3- pBS- PDS -S 14-pBS- PDS S colonies 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 7 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Mutation site as sequenced from transformed colonies 8 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Polymorphism in PDS introns indicate successful transformation 9 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

pBS- PDS as a versatile tool for nuclear transformation  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. 10 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Insertion of additional gene (psaD- ble cassette was driven from the pGenD- ble of Fischer and Rochaix, 2001) 11 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

ble gene is present in transformed colonies 1-5- pBS- PDS -L- ble 3 ’ 18, 19- pBS- PDS -S- ble 5 ’/ 3 ’, respectively colonies 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 12 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Insertion of ble via pBS- PDS-ble  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. 13 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Vector for Chloroplast Transformation pUC- atpX-aadA was kindly provided by (Goldschmidt-Clermont et al., 1991) based on the method presented in (Gutiérrez et al., 2012). 14 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

aadA gene is present in transformed colonies 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 15 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

pUC- atpX-aadA -16S-23S is suitable for chloroplast transformation  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. 16 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Transformation results Vector Bombarded Resistant Frequency of Average (per plate) introduced cells (*10 6 ) colonies* resistant cells (*10 -6 ) +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 - 6 20 3.3 5 ± 3 16S-23S 17 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Conclusions  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 . 18 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Concluding Remarks  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. 19 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013

Thank you!!!! 20 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 16/09/2013