A pair of specific primers derived from Arthrobotrys oligospora Serine Protease P186 gene in GenBank was designed and used to amplify P186 gene by RT-PCR from total RNA extracted from Arthrobotrys oligospora.The RT-PCR product was cloned into the expression vector pET32a to generate,recombinant pET32a-P186,and pET32a-P186 plasmid was transformed into E.coli BL21(DE3)and induced by IPTG.Sequence analysis demonstrated that P186 cDNA contained a insert of 1 224 bp encoding 407 amino acids,Among them 1-21 for the signal peptide sequence,amino acid sequence of 156-167,192-202,343-353 were aspartic acid(Asp160),histidine(His192)and serine(Ser345)active site area,respectively,which are belonging to the family of Subtilase.The amino acid also contained two potential N-terminal glycosyauon sites(Asn249,Asn342) and a substrate binding region of S1(SBP)Ser251Ile252Gly253 and Ala277Ala278Gly279.SDS-PAGE analysis showed that the product had a molecular weight of about 62 kDa.Western Blot analysis indicated that the recombinant protein could specifically react with polyclonal antibody against protein crude extracts.In order to reveal the molecular mechanism of Arthrobotrys oligospora predatory nematodes,In this study,the serine protease P186 gene of Arthrobotrys oligospora was firstly cloned and expressed,which laid a foundation of further study on biological function of P186.
ZHAO Hai-long
,
MENG Qing-ling
,
QIAO Jun
,
CHEN Shuang-qing
,
WANG Guo-chao
,
XIE Kun
,
HU Zheng-xiang
,
CAI Xue-peng
. Cloning and Prokaryotic Expression of Serine Protease P186 Gene of Arthrobotrys oligospora[J]. Acta Agriculturae Boreali-Sinica, 2014
, 29(4)
: 93
-97
.
DOI: 10.7668/hbnxb.2014.04.015
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