This part consists of three basic parts that can be directly linked to the fungal expression vector which can express in our fungus.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 903
Illegal NheI site found at 2993
- 21Illegal BglII site found at 137
Illegal BamHI site found at 3059
Illegal XhoI site found at 693
Illegal XhoI site found at 1075
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 2553
Illegal NgoMIV site found at 3695
Illegal AgeI site found at 1282
Illegal AgeI site found at 1430
- 1000Illegal BsaI.rc site found at 1109
Illegal BsaI.rc site found at 3409
The HsbA from Beauveria bassiana encodes a kind of membrane surface hydrophobic protein which helps our spores adhere to the wax on the cockroach body surface. Moreover, with the overexpression of HsbA, our spores can more effectively adhere to the cockroach. Then it will follow as spores’ germination, germinal tube, appressorium and the next penetrating process.
This part was inserted into the expression vector by restriction sites EcoRI and PstI (Fig.1), and the correct construction of this recombinant plasmid was confirmed by PCR identification and sequencing of the PCR products.
We transformed the expression vectors into Metarhizium anisopliae 128 by the method of Xiaoling Wang, and the positive clone was confirmed by G418 sulfate screening and nucleic acid electrophoresis.(Fig.2)
The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total protein by FastPrep and ultrasonic crushing. The lysate was then centrifuged and the supernate was electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining.(Fig.3)
Besides, by using the method of compared four areas of each wing in the scanning electron microscope before and after treatment which is ‘put the petri dishes on WD-9405B horizontal shaking table and opened the lowest rolling speed for 10min (to make the spores on the wings evenly impacted by the water flow)’ in the HsbA macro verification protocol, we can finally compared whether there was any change in the position and number of spores in the observing area. (illustrated with Fig.4 and Fig.5)
It’s obvious that it happens great change（circle in red）in the position and number of spores in the observing area of wild-type Metarhizium anisopliae 128 experimental groups.
It’s obvious that it almost happens no change except the place (circled in red) in the position and number of spores in the observing area of Metarhizium anisopliae HsbA transformant experimental groups.
Finally, the following chart (Fig.6) can be obtained by statistical data of four areas in all experimental groups.
In conclusion, this result well confirmed that Metarhizium anisopliae HsbA transformant certainly enhanced the capacity of adhesion.