Cloning of Insecticidal BT Genes for Application to Protection of Mushrooms from Fungus Gnats

Open Access
Author:
Curtis, Matthew Steven
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Ming Tien, Honors Advisor
  • Wayne Roger Curtis, Thesis Supervisor
  • David Scott Gilmour, Faculty Reader
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • Bacillus thuringiensis
  • BT
  • Molecular Cloning
  • Agaricus bisporus
  • Mushrooms
  • Biocontrol
Abstract:
This thesis describes the initiation of a project to generate mushrooms that will be protected from pests using the insecticidal proteins of the bacterial Bacillus thuringiensis (BT). The concept builds on the discovery of a previous student that found that proteins were transported from the lower mycelial layer up into the mushroom cap in such a way that the resulting edible mushroom is only contains the insecticidal proteins and does not contain the bacterial genes. This is accomplished by generating a BT-transgenic mycelium to be used as the lower under-layer, with a non-transgenic over-layer that generates the edible mushroom cap. The thesis work started with learning the basics of molecular biology for gene cloning and mushroom transformation, while conducting the background research on the BT insecticidal proteins as well as the target insect pests. The pests are ‘fungus gnats’ where their larval ‘maggot’ stage feeds on the mycelium within the compost. It is this larval stage that the BT can kill very selectively due to the behavior of the BT proteins that are in a crystal form until the dissolve in the alkaline gut of the larva to be active. This same behavior is why these proteins are non-toxic to humans and used in many GMO crops. This discovery phase of the research was conducted largely independently, since this project was not a part of an existing research project. The effort revealed the wide array of BT proteins where there is not only many BT genes in a given strain of B. thuringiensis, but there are many different strains - each of which has different specificity for killing larva of different insects. The literature review revealed that Bacillus thuringiensis serovar Israelensis was a good candidate for BT genes. The experimental component of the thesis involved obtaining these strain and the subsequent cloning of eight genes from its native megaplasmid. The genes were each PCR amplified with individually designed primers that included restriction fragment ends to facilitate subsequent DNA manipulation. The PCR product was digested and ligated into the PET28C cloning vector which was placed into two E.coli strains. The high-copy number E.coli DH5α was used for sequence verification; the E.coli BL21 is an expression vector that will be used to test the expressed proteins on fungus gnat larva to determine the relative toxicity of the various BT gene candidates. As part of training for this project, transformation methods for Agaricus bisporus (common supermarket button mushroom) were learned to transform reporter genes into this mushroom using hypertranslation sequences. As a result of this aggregate of general methods and specific molecular biology, the stage has been set to conduct the BT bioassay and generate the transgenic mushrooms, where the final stage of the thesis has been 'passing the torch' to a freshmen honors student who will continue towards the goal of generating BT protected mushrooms which do not contain the transgene and therefore provide for gene containment as well as the very interesting question of whether the mushrooms are a GMO food or not - since they do not contain the transgene (the current paradigm of GMO crops).