GLU Gene From Chaetomium Globosum: Cloning, Expression And Characterization

In agricultural production, it is always important to control plant pathogens, especially pathogenic fungi. At present, mainly chemical pesticides are used in the control of most pathogenic fungi. However, the use of chemical pesticides can lead to pollution of the environment and damage the health of human beings as well as livestock. The use of biocontrol for the inhibition of plant pathogens should be taken into consideration. Chaetomium globosum produces some cell wall hydrolases includingβ-1, 3-glucanase which can damage the cell walls of plant pathogenic fungi. Therefore, the fungus can contribute to biocontrol. As a result, C. globosum can be used in the search of molecular mechanisms and novel genes which can be applied to biocontrol.In order to produce biopesticides from a recombinantβ-1, 3-glucanase, Pichia pastoris expression system was used in this research, taking advantage of its high expression of extrinsic proteins and ease of cultivation. Initially, BLASX was performed to search the 5'and 3'sequences ofβ-1,3-glucanase(glu) from the C. globosum EST database, which had already been constructed. Then, the novel gene glu was cloned from C. globosum by PCR with appropriate primers designed according to their EST sequences and the gene sequence was submitted to the GenBank database with the accession number FJ587923. Next, RNA was extracted from C. globosum induced by R. solani and the cDNA of glu was obtained after reverse transcription and PCR. After that, the recombinant plasmid pPIC9K-glu was constructed successfully by ligating the cDNA and the vector pPIC9K which were both restricted using EcoR I and Not I.Electrotransformation into P. pastoris GS115 was performed after the recombinant plasmid pPIC9K-glu and pPIC9K control were linearized with BspEⅠ. The tranformants were screened using clone PCR and two positive transformants were obtained. Then, SDS-PAGE and activity assay were conducted to obtain one transformant which could highly express the recombinantβ-1, 3-glucanase (GLU).The expressed GLU protein was characterized and its inhibition effect on six plant pathogenic fungi was studied. The result showed that the GLU was produced most abundantly until the 3rd day of induction and its optimal temperature was determined to be 45℃. Based on these conditions, the highest activity of GLU was 1.31U using laminarin as substrate. In addition, the activity of recombinant GLU was determined using the cell walls from F. oxysporum, R. solani, P. grisea, S. sclerotiorum, S. tritici and V. sordida as substrates respectively. The results showed that the activities were higher using the cell walls from F. oxysporum, R. solani, P. grisea as substrates, whilst they were lower using the cell walls from S. sclerotiorum, S. tritici, V. sordida as substrates. It demonstrates that GLU can more drastically hydrolyze the cell walls from F. oxysporum, R. solani and P. grisea, and a conclusion can be drawn that GLU can inhibit the growth of F. oxysporum, R. solani and P. grisea more effectively.In conclusion, the recombinantβ-1, 3-glucanase, which was produced by the transformed P. pastoris GS115, showed high activity. And it could inhibit some plant pathogenic fungi obviously. Therefore, this study can contribute to biocontrol both theoretically and practically.