关键词: 捻转血矛线虫, 血红素, Hc-hrg-2, 酿酒酵母, Δhem1敲除株, 异源表达, 拯救

Abstract:

【Objective】In previous study, we have identified a heme responsive gene Hc-hrg-2 in Haemonchus contortus (H. contortus), with a high transcriptional level in the presence of high concentration of heme. However its function in heme regulation is still lacking research. To verify that Hc-hrg-2 was involved in the intracellular heme transport, a hem1 gene knockout strain of Saccharomyces cerevisiae, which was heme deficient, was constructed by homologous recombination technique and then exogenously expressed Hc-hrg-2 of H. contortus to rescue the growth of the knockout strain. 【Method】The genomic DNA of S. cerevisiae BY4741 was used as a template to obtain the upstream and downstream homology sequences of hem1 (SGD: S000002640) gene. The plasmid pYES2-CT was used to obtain the screening marker URA3 sequence. Two overlapping PCR techniques were used to sequentially connect upstream homology sequence, URA3, and downstream homology sequence to form the knockout components which was purified and then transformed into BY4741 competent cells by lithium acetate transformation method, and the transformants were selected on SD /-URA plates supplemented with 250 μmol·L ^-1 5-aminolevulinic acid (ALA). PCR identification using multiple primer pairs was further performed to verify the correctness of Δhem1 strain. The Hc-hrg-2 sequence (GenBank: MK371241) of Zhejiang strain and its functional domain deleted sequence Hc-hrg-2(Δgst-n) and Hc-hrg-2(Δgst-c) were amplified from the plasmids and inserted into the yeast expression vector pESC-LEU through a seamless cloning kit. The expression vectors, which were identified and sequenced to be correct, were then transformed into Δhem1 competent cells and selected on SD/-URA/-LEU (containing 250 μmol·L ^-1 ALA) plates. PCR identification was performed to verify the positive exogenous expression strain. By comparing the growth of Δhem1 strain and its exogenous expression strains in SD/-URA/-LEU liquid medium with or without 250 μmol·L ^-1 ALA, the phenotype of the knockout strain were further verified and the effects of expression vectors on phenotype were excluded. The exogenous expression strains were induced by 2% w/v galactose and then sonicated to identify the protein expression by Western Blot. The induced strains were resuspended to an OD₆₀₀ of 0.2 and 4 μL of 5-fold serial dilutions of each induced strain was spotted onto 2% w/v galactose plates supplemented with either 250 μmol·L^-1 ALA or different concentrations of heme for 2 to 3 days at 28 ℃ to compare the growth of the strains. 【Result】The hem1 gene knockout strain was successfully obtained. Compared with wild strain, Δhem1 cannot synthesis heme in vivo and requires ALA (250 μmol·L ^-1) or heme (≥10 μmol·L^-1) for growth. The phenotypes of the exogenous expression strains were consistent with that of the knockout strain. Western Blot results indicated that 2% w/v galactose could successfully induce the expression of Hc-hrg-2 and its functional domain deleted gene in the knockout strain. Hc-hrg-2 expression allowed S. cerevisiae to import heme from the environment, and rescued the growth of Δhem1 strain. Notably, the deletion of two signature domains of Hc-hrg-2, a thioredoxinlike (GST-N) and a glutathione S-transferase C-terminal domain-like (GST-C), could reduce the effect of rescue. 【Conclusion】Hc-hrg-2 could facilitate heme uptake in cells and its functional domains, GST-N and GST-C, played an important role in this process. This study laid a solid foundation for further exploring heme transport mechanism of H. contortus.

Key words: Haemonchus contortus, heme, Hc-hrg-2, Saccharomyces cerevisiae, Δhem1 strain, exogenously expression, rescue