Cloning and Expression of Chitinase Gene AO-190 of Arthrobotrys oligospora XJ-A1 and Analysis of the Chitinase Activity of Recombinant Protein

doi:10.7668/hbnxb.2018.04.010

Abstract

Abstract: Nematode-trapping fungi can secrete chitinase in the process of infecting nematodes.In order to study the functions and molecular characteristics of chitinase, chitinase gene AO-190 of Arthrobo trys oligospora XJ-A1 isolate was cloned, analyzed and expressed in Escherichia coli by constructing prokaryotic expression vector pET32a-AO-190. The prokaryotic expression recombinant fusion protein was purified by Ni column and the chitinase activity was purified by chitinase ELISA kit.The results showed chitinase AO-190 gene had a length of 1 574 bp and four intron sequences. The sequence contained one open reading frame (ORF)with 1 251 bp that encoded 416 amino acids.The homology of the chitinase AO-190 gene sequence was 93.33% and the amino acid sequence was 92.55% respectively with Arthrobotrys oligospora standard strain (ATCC 24927)chitinase AO-190. There was a signal peptide, a Arginine-rich region profile, a Bipartite nuclear localization signal, a N-glycosylation site, a Protein kinase C phosphorylation site, a N-myristoylation site, a Casein kinase Ⅱ Amidation site and a Low-Complexity region.The proteins belonged to the family 18 glycoside hydrolase with prevalent conserved substrate binding domains SLGG and catalytic domains VDGVDLDLE.The major structural elements of secondary structure included random coils, alpha helix and extended strand while a typical (α/β)₈ phosphorylation site, a rounded bucket structure were predicted about tertiary structure.Phylogenetic analysis showed that the phylogenetic relationship of chitinase AO-190 was more close to the chitinase (EPS43772.1)originate from Dactylellina haptotyla who could produce short handle sticky ball and the chitinase (EWC46603.1) originate from Drechslerella stenobrocha who could produce constricting rings and had a significant evolutionary distance with chitinase of insects and vertebrates.The recombinant protein was identified by SDS-PAGE analysis that showed it had a molecular weight of about 63 ku, which was consistent with prediction. Western Blot analysis indicated that the recombinant protein could specifically react with polyclonal antibody against protein crude extracts.The purified recombinant chitinase activity was 222 IU/L that was measured using by chitinase ELISA kit.

Key words: Arthrobotrys oligospora, Chitinase, Prokaryotic expression, Protein purified, Chitinase activity

CLC Number:

Q78
S432.4

GONG Shasha, MENG Qingling, QIAO Jun, ZHONG Wenqiang, HUANG Yunfu, ZHANG Guowu, CHEN Ying, CAI Xuepeng. Cloning and Expression of Chitinase Gene AO-190 of Arthrobotrys oligospora XJ-A1 and Analysis of the Chitinase Activity of Recombinant Protein[J]. Acta Agriculturae Boreali-Sinica, 2018, 33(4): 67-74. doi: 10.7668/hbnxb.2018.04.010.

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