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Application and verification of auxin-inducible degron system in Toxoplasma gondii Chinese type 1 strain
Received date: 2019-10-24
Online published: 2020-05-11
Supported by
Supported by the National Natural Science Foundation of China(81071375);Beijing Natural Science Foundation(7182023)
Objective To regulate the expression of fluorescent proteins in Toxoplasma gondii Chinese type 1 strain using the auxin-inducible degron (AID) system.Methods The nucleotide sequence of plant auxin receptor-transport inhibitor response protein 1 (TIR1) fused with a FLAG tag at the 3′ end (TIR1-FLAG) was synthesized, and ligated into the pTub-eGFP-CAT vector to obtain the pTub-TIR1-FLAG-CAT expression plasmid. The plasmid was transfected into the Toxoplasma gondii Chinese type 1 strain by electroporation. After chloramphenicol screening and limiting dilution, positive clones were selected, in which the inserted TIR1 gene and its expression was examined by PCR and Western blotting. The verified strain clone with correct insertion was designated as the TIR1 strain. The plasmid pTub-mCherry-Ty-AID-DHFR expressing the mCherry-Ty-AID reporter gene was constructed and integrated into the genome of the TIR1 strain by electroporation, from which a clone capable of stable expression of mCherry-AID protein was obtained by pyrimethamine screening and ultimate limiting dilution, and designated as TIRI:mCherry-AID strain. The TIR1:mCherry-AID parasites were divided into the regulating group and control group, and cultured with an auxin, indole-3-acetic acid (IAA) solution (500 μmol/L) or an equal volume of ethanol for 3 h accordingly. Then the expression of mCherry fluorescent protein was detected by indirect immunofluorescence assay (IFA) and Western blotting using anti-TgGAP45 and anti-Ty-1 antibodies.Results The PCR amplification showed a specific amplification band at 1 816 bp. Western blotting showed that the anti-FLAG-Tag antibody can recognize a protein band with a relative molecular mass (Mr) of 70 000 in the TIR1 strain, and anti-Ty-1 antibody can detect a specific band with Mr of about 58 000 in the TIR1:mCherry-AID strain, which equales to the theoretical size of mCherry-Ty protein fused with AID at the C terminus; however, no specific band was detected in the auxin/IAA group. IFA assay indicated that red fluorescent protein was seen in the TIR1:mCherry-AID in the control group, but no mCherry protein was detected in the auxin/IAA group.Conclusion The AID system can regulate mCherry fluorescent protein expression in T. gondii Chinses 1 strain.
Ai-xia YAN , Yong-gen JIA , Yang ZOU , Jing-jing LI , Min-jun HUANG , Jun-chao GU . Application and verification of auxin-inducible degron system in Toxoplasma gondii Chinese type 1 strain[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(2) : 207 -212 . DOI: 10.12140/j.issn.1000-7423.2020.02.012
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