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Production of antibodies for the screening of transgenic plants individually expressing phbA, phbB and phbC
Author
Loo, Hui Phing
Supervisor
Gan-Yap, Yik Yuen
Abstract
Polyhydroxybutyric acid (PHB) is a carbon storage material synthesised by many microorganisms under nutrient limiting conditions. PHB has properties resembling that of conventional plastics such as polypropylene, thus it is regarded as an ideal candidate to replace conventional plastics and rectify problems arising from plastic waste disposal. PHB is synthesised from acetyl-CoA by the consecutive action of 3-ketothiolase, acetoacetyl-CoA reductase and PHB synthase. Currently PHB is commercially produced from bacterial fermentation processes and marketed by Zeneca Bio Products (formerly known as ICI). The coding sequences of phb genes have been cloned into recombinant E. Coli strains. However, this method of production is not as cost effective as compared to using a plant system.
Molecular biologists have been successful in engineering transgenic Arabidopsis thaliana, rapeseed, cotton, maize and soybean to produce PHB. This thesis seeks to engineer three host plants each harbouring at least one of the PHB enzymes by Agrobacterium-mediated transformation. A long-term goal is to acquire a single hybrid transgenic plant expressing all three functional enzymes, for the production of PHB in the plastid. This can be achieved by particle bombardment or by cross-pollination of individual plants harbouring at least one of the phb genes. Gene constructs containing individual phb genes downstream of a transit peptide would target the expression of those genes to within the plastid. The plastid was selected because an abundance of acetyl-CoA is present in that compartment. A number of plant species (African violet, Balsam, Celosia, Gloxinia and Petunia) were experimented to select an ideal plant species for transformation. Three plant species (African violet, Gloxinia and Petunia) were chosen for transformation by particle bombardment and Agrobacterium transfer because of their established regeneration system and the ease to perform cross-pollination.
In order to identify transgenic plants harbouring the phb genes, there was a necessity to generate antibodies. Coding sequences of the phb genes were introduced into the pMAL and pQE bacterial expression systems. Three pMAL constructs (pMAL-Thio, pMAL-Red and pMAL-Syn) were generated. Bacterially expressed fusion proteins were affinity purified and used as antigens for rabbit immunisation. Anti-Thio, anti-Red and anti-Syn antibodies were successfully generated and verified by immunoassays. The presence of the transferred genes was determined by PCR screening whereby 12 transgenic Petunia and 9 transgenic African violet were tested positive. The slow growth of African violet only allowed for PCR screening to be carried out. Further analysis of the 12 transgenic Petunia by dot blot hybridisation using different DNA probes showed that the phb genes have been integrated and transcribed in eight plant lines. Plant proteins from 11 Petunia lines were assayed for the presence of the PHB enzymes by Western blotting of which two plant lines expressed 3-ketothiolase, three expressed acetoacetyl-CoA reductase and two expressed PHB synthase.
Cross-pollination of Petunia plants to obtain a hybrid containing at least two of the phb genes is in progress. The long-term goal of acquiring a single Petunia hybrid with all of the three phb genes looks promising despite the fact that a lower PHB yield will be expected. Nevertheless, using Petunia as a model system will enable one to have a better understanding of PHB production in non-oil producing plant.
Molecular biologists have been successful in engineering transgenic Arabidopsis thaliana, rapeseed, cotton, maize and soybean to produce PHB. This thesis seeks to engineer three host plants each harbouring at least one of the PHB enzymes by Agrobacterium-mediated transformation. A long-term goal is to acquire a single hybrid transgenic plant expressing all three functional enzymes, for the production of PHB in the plastid. This can be achieved by particle bombardment or by cross-pollination of individual plants harbouring at least one of the phb genes. Gene constructs containing individual phb genes downstream of a transit peptide would target the expression of those genes to within the plastid. The plastid was selected because an abundance of acetyl-CoA is present in that compartment. A number of plant species (African violet, Balsam, Celosia, Gloxinia and Petunia) were experimented to select an ideal plant species for transformation. Three plant species (African violet, Gloxinia and Petunia) were chosen for transformation by particle bombardment and Agrobacterium transfer because of their established regeneration system and the ease to perform cross-pollination.
In order to identify transgenic plants harbouring the phb genes, there was a necessity to generate antibodies. Coding sequences of the phb genes were introduced into the pMAL and pQE bacterial expression systems. Three pMAL constructs (pMAL-Thio, pMAL-Red and pMAL-Syn) were generated. Bacterially expressed fusion proteins were affinity purified and used as antigens for rabbit immunisation. Anti-Thio, anti-Red and anti-Syn antibodies were successfully generated and verified by immunoassays. The presence of the transferred genes was determined by PCR screening whereby 12 transgenic Petunia and 9 transgenic African violet were tested positive. The slow growth of African violet only allowed for PCR screening to be carried out. Further analysis of the 12 transgenic Petunia by dot blot hybridisation using different DNA probes showed that the phb genes have been integrated and transcribed in eight plant lines. Plant proteins from 11 Petunia lines were assayed for the presence of the PHB enzymes by Western blotting of which two plant lines expressed 3-ketothiolase, three expressed acetoacetyl-CoA reductase and two expressed PHB synthase.
Cross-pollination of Petunia plants to obtain a hybrid containing at least two of the phb genes is in progress. The long-term goal of acquiring a single Petunia hybrid with all of the three phb genes looks promising despite the fact that a lower PHB yield will be expected. Nevertheless, using Petunia as a model system will enable one to have a better understanding of PHB production in non-oil producing plant.
Date Issued
1999
Call Number
SB123.57 Loo
Date Submitted
1999