摘要
【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,
【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 ef
作者
周静茹
吴飞
陈学秋
黄艳
时恒枝
杜爱芳
杨怡
ZHOU JingRu;WU Fei;CHEN XueQiu;HUANG Yan;SHI HengZhi;DU AiFang;YANG Yi(College of Animal Science,Zhejiang University/Key Laboratory of Animal Preventive Medicine of Zhejiang Province,Hangzhou 310058)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第8期1795-1804,共10页
Scientia Agricultura Sinica
基金
国家自然科学基金(31602041)
国家重点研发计划(2017YFD0501200)
浙江省基础公益研究计划(LGN20C180005)。
