Bbchit_Beauveria bassiana ARSEF 2860
This part is the coding sequence (CDS) of Chitinase from The Beauveria bassiana ARSEF 2860(GenBank Acc.No.txid655819). It can encode chitinase and effectively decomposes chitin. We transferred this gene to enhance the penetration of Metarhizium anisopliae.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NotI site found at 581
- 21Illegal XhoI site found at 568
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 250
Illegal NgoMIV site found at 779
- 1000COMPATIBLE WITH RFC
In order to make Metarhizium anisopliae penetrate the corpus callosum more efficiently, we constructed an expression vector containing part Bbchit(Fig.1)
We constructed a shuttle vector to transform this part and the positive clone was confirmed by G418 sulfate screening and nucleic acid electrophoresis. (Fig.2)
The crude enzyme solution was obtained by cell disruption using ultrasonic, followed by SDS-PAGE protein electrophoresis and Coomassie blue staining. (Fig.3)
To determine the activity of chitinase, we improved it according to the DNS colorimetric method of Kan Zhuo, Xiaozhen Shi. First, the standard curve was drawn with different concentration gradients of glucose solution, and 0.5 ml of the wild-type and transformed type 1, 3, 5, 7, 9 and 12 days of culture solution were respectively taken for enzyme activity test: the crude enzyme obtained after filtering the culture solution was used. The solution was mixed with 0.5 ml of 1% chitin colloid, reacted at 37℃ for 60 min, and then added to a 0.5 ml DNS boiling water bath for 10 min. The absorbance of the obtained product was measured and the enzyme activity was calculated. There were three groups of wild-type and transformed type. Parallel, three parallel experiments were performed in each group, and the final data were averaged. We calculate activity based on the standard curve formula: U=（A540+0.03279）/2.202(Fig.4), a summary of the data at different times is made into a line chart as follows. We can see that after 12 days the transformed type’s enzyme activity is still growing and the wild-type is falling. (Fig.5)
In order to verify the function of Bbchit from a macro level, we improved Kan Zhuo's chitin transparent circle method for verification. We stained the czapek solid medium without chitin colloids in red with 0.1% Congo red dye solution and then cultured wild-type and transformed Metarhizium. Compared with the size of the colony, the size of the transparent circle was compared to obtain the transformed type. The size of the transparent circle is the diameter of the chitin transparent ring (R2) and colonies. The ratio of the diameter (R1), expressed as R2/R1.The conclusion that the chitinase activity of Metarhizium anisopliae is enhanced.(Fig.6)
Therefore, these results well confirmed that the chitinase activity of Metarhizium anisopliae Bbchit transformant is about 1.3 times that of wild-type Metarhizium anisopliae 128. Our modified fungus certainly enhanced the capacity of penetration.