Construction of plasmid vector
We used the fungal plasmid pBC (donated from professor Xie) for our project, which can propagate in Metarhizium anisopliae 128. The vector contains the following parts:
|PgpdA||PgpdA is the constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans.|
|TtrpC||TtrpC is a tryptophan terminator from Aspergillus nidulans.|
|HsbA||Encoded by the gene HsbA from Beauveria bassiana ARSEF 2860.|
|Bbchit||Encoded by the gene Bbchit from Beauveria bassiana ARSEF 2860.|
|MCL1||MCL1_Metarhizium robertsii ARSEF 23|
|Tryptophan-MazF||Suicide switch, induces apoptosis in the absence of tryptophan.|
Inside the vectors，there are four gene pathways. PgpdA promoter starts the transcription of HsbA, Bbchit, MCL1, Ttyptophan-MazF. And TtrpC terminator terminates the transcription. It is well to be reminded that the pathway, PgpdA-Ttyptophan-MazF-TtrpC has a special switch, which induces apoptosis in the absence of tryptophan.
The model organism used in the project is Metarhizium anisopliae 128 (presented by professor Zhongkang Wang from Chongqing University), which is a major strain of Metarhizium anisopliae 128 with strong virulence.
Transformation and expression
We used the transformation method of Xiaoling Wang to transform Metarhizium anisopliae 128, and obtained stable transformant through the screening of G418 sulfate, and then verified the successful transformation through extracting genome, primer PCR and nucleic acid electrophoresis.
To verify the existence and function of HsbA protein, the following treatment is done.
In order to verify the function of the transferred chitinase, the following experiments were performed.
In order to verify the ability of immune-avoidance in MCL1-transformant, the following experiments were performed.
To verify the function of Suicide Switch, and get the limited concentration of Tryptophan, the following experiments were done:
Application and realization
In order to make our Metarhizium anisopliae spores into products, we designed GreenGround, a containing box for spores, with a structure similar to the cockroach house.
Compared to regular chemicals, GreenGround reduces the elimination of cockroaches. To prove its usefulness, we built a simulated room to test it. But according to the principle of no release, we could not use the modified Metarhizium anisopliae. So in the experiment we used the wild type Metarhizium anisopliae.
We built two mini-houses using KT Board (made of polystyrene) to simulate two rooms, which connected with a corridor. We design control group without any drugs, and experimental group with cockroach killing chalk, BAYER-Premise and out product equipped with M.anisopliae emulsifiable powder. Then, we put thirty cockroaches into one room each time, and came back to see the results after three days. We focused more attention on migration rate and mortality.
Finally, we got results that the migration rate of control group was 7.78%, while 21.11%, 20.00%, 6.67% respectively in groups using cockroach killing chalk, BAYER-Premise and our product. For mortality in three days, 77.78% in group using chalk, 84.44% in group using Premise, 28.89% in group using our product, and 2.01% in group using nothing. Statistical Analysis in our model shows our product with M.anisopliae emulsifiable powder will not cause a high migration rate of cockroaches comparing with traditional products, although it has lower mortality within three days.
What’s more, we tested the mortality between groups using and without using our box to equipped M.anisopliae emulsifiable powder. The mortality in group using our box was 34.14%, while 12.22% in group without using any box. The result show that that it is necessary for us to use our Green-Ground box to hold M.anisopliae emulsifiable powder.
|Migration Rate||Mortality||Gnawing Rate|
|Cockroach killing chalk||21.11%||77.78%||2.22%|