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Study on Characteristics and Expression Regulation Elements of Chitinase from Bacillus Licheniformis MY75

Author: XiaoLiang
Tutor: ChenYueHua；CaiJun；CaiBaoLi
School: Nankai University
Course: Microbiology
Keywords: Bacillus licheniformis Antifungal activity Promoter Repetitive sequences Missing Regulation
CLC: Q936
Type: PhD thesis
Year: 2010
Downloads: 166
Quote: 3
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Abstract

The MY75 strain is a Gram-positive Bacillus preserved in this room. When chitin medium, the strain can be abundantly expressed extracellular chitinase nearly 1000 Bacillus screening, and enzyme activity is highest. In order to clarify the status of the MY75 strain classification, first identified this strain. Was found in the identification process, MY75 strain 16S RNA sequence and Bacillus subtilis Bacillus flora (Bacillus subtilis group) having a high degree of similarity between, but can not use the 16S RNA sequence analysis method is now widely used to effectively distinguish . Thus, again another two protein coding genes gyrA (DNA encoding the A subunit of gyrase, gyrase subunit A) and the rpoB (p subunit of DNA encoding the RNA polymerase mediated, RNA polymerase subunit beta) sequence as the basis, and reference Biolog Microbial Identification results finalized MY75 strains the licheniformis bacillus (Bacillus lichenifromis,) MY75 strain produced after 96 hours of incubation, extracellular chitinase peaked -4.645 U / mL culture supernatant from bacteria The prepared crude protein solution fungistatically experiments, found that strains of crude protein after induction of chitinase to completely suppress wheat Gibberella (Gibberella saubinetii) and the Aspergillus niger spore germination (Aspergillus niger), and the uninduced sample Without this effect, preliminary evidence of the antifungal activity in the the MY75 strain culture supernatant derived from its high amount of expression of extracellular chitinase. Preliminary MY75 strains in the culture supernatant of crude protein separation and purification, and found that a specific protein band of approximately 55 kDa in size showed chitinase activity detected by zymography. This protein, the time of flight mass spectrometer (time-of-Flight mass spectrometer, TOF MS) analysis, found chitinase homology in its amino acid sequence and the protein database of the size of 67 kDa of the Bacillus licheniformis. Amplified and cloned the chitinase gene chiMY75 MY75 strains has been submitted to GenBank, Bacillus licheniformis chitinase primers were designed. The open reading frame of the gene (Open Reading Frame from the length of the ORF) of 1797 bp, encoding 599 amino acids of the predicted protein size of 67 kDa. The Bacillus licheniformis already in the ORF sequences in GenBank chitinase gene sequence similarity of 99%. ChiMY75 subcloned into the expression vector pET28a was transformed into E. coli. Detected in the transformant cells, crushing the supernatant to a specific expression of the protein of 67 kDa in size. The heterologous expression chitinase ChiMY75, characteristics of the study. The best role of the enzyme at pH 7.0, the best role in a temperature of 50 ° C. Manganese, chromium, zinc, silver four ions can significantly reduce the activity of ChiMY75; lithium, sodium, and magnesium ions on the enzyme activity has a minor role in promoting; both copper and iron ions cause the complete loss of activity of the protein. Antifungal activity, the enzyme activity of 6.32 U / the mL of ChiMY75 be able to completely inhibit spore germination of of gibberellic wheat and Aspergillus niger. Insecticidal bioassay, the Bacillus thuringiensis the addition of ChiMY75 makes the beet armyworm (Spodoptera exigua) larvae virulence improve the 27%. Order to study the regulatory elements of the Bacillus chitinase, cloning the MY75 strains chitinase gene complete sequence chiMY75full, including the ORF and its upstream regulatory region of 432 bp. While Our previous clone 15A3 strain of Bacillus thuringiensis chitinase the complete gene chiB also as an object of study. Above two chitinase gene upstream regulatory sequences were analyzed using software to predict the potential promoter sequence and other regulatory elements. The sequence analysis revealed that both the upstream sequences are present in both the length of 16 bp direct repeats. Two complete genes can carry their own promoter the chitinase abundantly expressed in E. coli. Repeat sequences found were designated part of and all missing from the three levels of activity, protein expression, as well as the amount of specific mRNA transcripts observed changes in the missing gene regulation expression. Enzyme Activity test results prove, deletion the chi gene transformant culture solution of the enzyme activity was significantly higher than the full chi gene transformants forward repeat sequence; protein SDS-PAGE electrophoresis showed that the deletion forward a series of chi gene repeat sequence The expression of chitinase protein was significantly higher than the complete CHI gene transformant; Northern Blot results show that, after deletion repeat sequence, the amount of mRNA transcripts corresponding to the two kinds of chitinase genes also significantly improved. These results demonstrate that the direct repeats of the chitinase gene of Bacillus is negative control gene expression regulatory elements, is likely to be a transcription inhibitor binding sites. Designated deletion results also show that the the missing all repeating the sequence chiB gene missing part of the repeat sequence chiB gene compared three detection levels are significantly higher. In the present study, we will MY75 strain was identified as Bacillus licheniformis. Heterologous expression in E. coli, to determine MY75 strains chitinase antifungal activity of Bacillus thuringiensis insecticidal synergism. Were found in the upstream of the chitinase gene encoding two Bacillus direct repeats, by direct repeats sentinel deletion, prove that the sequence is the negative regulatory element of chitinase gene expression in, at the transcriptional chitinase expression level control. In this study, not only the foundation for the study of the chitinase gene regulatory mechanism for Bacillus, also proved MY75 strains in biological control potential

Study on Characteristics and Expression Regulation Elements of Chitinase from Bacillus Licheniformis MY75

Abstract

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