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Cloning, Functional Analysis and Prokaryotic Expression of Biocontrol-Related Phy Gene in Serratia Proteamaculans 336X

Author: MaHaiZuo
Tutor: WangGang
School: Henan University
Course: Microbiology
Keywords: Serratia proteamaculans Bio-control mechanism Gene cloning Prokaryotic Express
CLC: S476.1
Type: Master's thesis
Year: 2011
Downloads: 19
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Abstract

Serratia proteamaculans 336X can against take-all disease of wheat, which is caused by Gaeumannomyces graminis var. tritici （Ggt）. It is a potential biological control agent. So it is important to research the biological control mechanisms of 336X against Ggt, and explore the interactions between PGPR and plants on the molecular level.At present, to amplify the ORF from a putative gene which was involved in ABC transport system, primers phyF/R were designed according to the genome sequence of Serratia proteamaculans 568. In our research, we obtained the full length of 1569 bp DNA fragment from 336X genomic DNA by PCR with the primers phyF/R. Then we cloned this DNA fragment into vector pMD-18T, and according the result of sequencing analysis, we found that the DNA fragment we obtained showed a high degree of homology to the putative 4’-phytas gene of Serratia proteamaculans 568 with identity 94%; so this fragment was named the phy gene. Conservative domain analysis also showed that the predicted amino acid sequence of the phy gene product had about 15-21% identity to the soluble periplasmic binding protein-like II（PBP2_NikA_DppA_OppA_like₇）. N-terminal 31 amino acids sequence of signal peptide was founded by signal peptide prediction program SignalP 3.0 Server analysis, it also predicted that mature peptide chain may be cut at 27-28 amino acids.In order to study the functions of the phy gene, an allelic exchange vector was constructed by cloning phy fragment into suicide vector pKAS32 and was interrupted with Km-anticipate fragment. The phy mutations were obtained by conjugation of pKAS32-phy-Km from E.coli S17-1 into 336X, three phy mutants were obtained and identified by PCR. After a series of examinations, we found that the mutants can’t uptake Ni2+ effectively, and this result suggests that the phy may be able to encode the Ni2+ binding protein involved in Ni2+ transport. In addition, we found that the mutants failed to suppress take-all caused by Ggt, and showed lower susceptibility to the exudates of roots than the wild-type; moreover, we detected the number of the mutants’colonies reduced that on the surface of wheat roots. These results may help us to explain the biocontrol mechanisms of 336X.To further characterize the phy gene, the ORF of phy was inserted into pET-30a to express recombinant protein in E.coli. BL21（DE3）. The expression of predicted 60 kD recombinant proteins was induced by isopropyl beta-D-thiogalacto-pyranoside（IPTG）, and the induced expression conditions was also optimized. The optimal inducing conditions were as following:add IPTG to medium with final concentration at 0.5mmol/L, and culture at 28℃for 5 h. The recombinant protein existed in a form of inclusion body in the cells and released by ultrasonic broken, dissolved with 6 M guanidine hydrochloride. The target protein was purified with Ni2+ -NTA affinity chromatography on denaturing conditions and renatured by gradient dialysis. This work is the foundation of researches as protein sequencing, active site analysis, and structure identification.

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