China Animal Husbandry and Veterinary Medicine ›› 2021, Vol. 48 ›› Issue (11): 4192-4203.doi: 10.16431/j.cnki.1671-7236.2021.11.029

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Analysis of Key Action Sites of Salmonella Typhimurium STM LT2 Hfq

DUAN Shiyu1,2, PAN Yong1,2, YANG Yang1,2, ZHANG Jiali1,2, LINGHU Yuanfeng1,2, YANG Qi1,2,3, ZHOU Bijun1,2,3   

  1. 1. College of Animal Science, Guizhou University, Guiyang 550025, China;
    2. Laboratory of Animal Diseases in Guizhou Province, Guiyang 550025, China;
    3. Key Laboratory of Animal Diseases and Veterinary Public Health of Guizhou Province, Guiyang 550025, China
  • Received:2021-05-10 Online:2021-11-20 Published:2021-11-01

PDF (PC)

29

Abstract: To explore the possible key action sites of Hfq, GcvB and their target gene oppA, in this study, λ-Red homologous recombination technology was used to construct mutant strains with core amino acids on the distal and proximal faces of Hfq and Hfq C-terminal truncated strains. On this basis, the corresponding Hfq-His6 protein tag fusion strains were constructed. P22 phage transduction technology was used to construct corresponding GcvB gene deletion strain and oppA::lacZ gene fusion strains. The protein level of Hfq was detected by Western blotting test, the transcription level of GcvB and oppA gene was detected by Real-time quantitative PCR, and the level of oppA protein was detected by β-galactosidase test. Western blotting test results showed that the mutation of core amino acids on the distal and proximal faces of Hfq and the truncation of the C-terminus of Hfq all increased the protein level of Hfq to varying degrees. The results of Real-time quantitative PCR showed that compared with control group, the mutation of the core amino acid of the proximal face of Hfq down-regulated the transcription level of the GcvB gene. The results of β-galactosidase test showed that compared with control group, the mutations of core amino acids on the distal and proximal faces of Hfq and the truncation of the C-terminus of Hfq did not cause significant changes in the transcription level of oppA gene, but they all increased the level of oppA protein to varying degrees. Among them, when the core amino acid mutation of Hfq proximal face and the C-terminus of Hfq were truncated to amino acid at positions 65 and 72, the protein level of oppA was up-regulated most obviously, up-regulating 2.9, 3.3 and 2.0 times, respectively. The results indicated that the proximal face of Hfq was very important for Hfq to maintain the stability of GcvB, the amino acid at position 56 of the proximal face of Hfq and the amino acid at positions 65 to 87 of the C-terminus of Hfq were negatively correlated with the level of oppA protein.

Key words: Salmonella Typhimurium; chaperone Hfq; λ-Red homologous recombination system; GcvB; oppA

CLC Number: