捻转血矛线虫Hc-hrg-2拯救血红素缺陷型酵母生长表型

Hc-hrg-2 of Haemonchus Contortus Rescues the Growth of Heme Deficient Yeast Strain

【目的】已有研究表明捻转血矛线虫(Haemonchus contortus)Hc-hrg-2属于血红素应答基因,高浓度血红素的刺激可导致该基因的转录水平上调,但其在血红素调控中的功能尚缺乏研究。研究通过同源重组技术构建血红素合成缺陷型酿酒酵母(Saccharomyces cerevisiae)hem1敲除株,通过异源表达Hc-hrg-2对该敲除株进行表型拯救试验,以期验证Hc-hrg-2参与细胞内血红素转运的功能。【方法】以S.cerevisiae BY4741基因组为模板,设计引物,扩增获得hem1(SGD:S000002640)基因序列5'和3'侧翼区同源臂;同时以pYES2-CT质粒为模板设计引物,扩增获得筛选标记URA3序列;利用两次重叠PCR技术依次将测序正确的上游同源臂、URA3、下游同源臂序列串联成基因敲除组件,并通过醇沉法对敲除组件进行纯化。采用醋酸锂转化法将纯化的敲除组件转化至BY4741感受态细胞中,经SD/-URA(含250μmol·L^(-1)5-氨基乙酰丙酸(ALA))培养基筛选,并利用多对引物进行PCR鉴定,以验证Δhem1敲除株的正确性。同时以含有H.contortus浙江株Hc-hrg-2序列(GenBank:MK371241)及其功能域缺失序列Hc-hrg-2(Δgst-n)和Hc-hrg-2(Δgst-c)的质粒为模板,利用特异性引物分别进行PCR扩增,通过无缝克隆将扩增得到的目的片段插入酵母pESC-LEU表达载体,经PCR鉴定以及测序验证后,采用醋酸锂转化法将正确的表达载体转化至Δhem1感受态细胞中,通过SD/-URA/-LEU(含250μmol·L^(-1)ALA)培养基筛选以及PCR鉴定验证阳性异源表达株的正确性。通过比较Δhem1敲除株及其各异源表达株在含有或不含有250μmol·L^(-1)ALA的SD/-URA/-LEU液体培养基中的生长情况,进一步验证敲除株的表型同时排除表达载体对表型的影响。用2%半乳糖对含有阳性表达质粒的敲除株进行诱导,取部分菌液进行超裂破碎,收集蛋白,通过Western Blot鉴定目的蛋白的表达;剩余菌体用去离子水重悬至OD600=0.2,用去离子水进行5倍比稀释,取4μL稀释后的菌液点至含有250μmol·L^(-1)ALA或者不同浓度血红素的诱导平板上,28℃培养2—3 d后比较敲除株的生长情况。【结果】成功获得hem1基因敲除株Δhem1,与野生株相比,Δhem1不能合成血红素,需外源添加ALA(250μmol·L^(-1))或血红素(≥10μmol·L^(-1))才能生长,且异源表达株和敲除株的表型一致。Western Blot结果表明2%半乳糖能诱导Hc-hrg-2及其功能域缺失基因在敲除株中成功表达,且表达Hc-hrg-2能够在低血红素浓度下(≤1μmol·L^(-1))促进酵母对血红素的摄取,拯救敲除株的生长缺陷,其硫氧还蛋白样结构域(GST-N)和谷胱甘肽S-转移酶C末端结构域(GST-C)的缺失会降低拯救的效果。【结论】捻转血矛线虫血红素应答基因Hc-hrg-2可促进细胞对血红素的摄取,其GST-N和GST-C功能域在该过程中发挥着重要作用,研究成果为后续深入研究捻转血矛线虫的血红素转运机制奠定基础。

【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 250μmol·L^(-1)5-aminolevulinic acid(ALA).PCR identification using multiple primer pairs was further performed to verify the correctness ofΔhem1 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 intoΔhem1 competent cells and selected on SD/-URA/-LEU(containing 250μmol·L^(-1)ALA)plates.PCR identification was performed to verify the positive exogenous expression strain.By comparing the growth ofΔhem1 strain and its exogenous expression strains in SD/-URA/-LEU liquid medium with or without 250μmol·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 4μL of 5-fold serial dilutions of each induced strain was spotted onto 2%w/v galactose plates supplemented with either 250μmol·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(250μmol·L^(-1))or heme(≥10μmol·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 ofΔhem1 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.