In yeast,ATG6 is a core molecule of the phosphatidylinositol 3-kinase complex I,which can affect the formation of autophagosome.To investigate the function of the T.gondii orthologue of ATG6 (TgATG6),relationships of TgATG6 and other ATG6 orthologues was analyzed through phylogenetic analysis.The subcellular localization of TgATG6 was detected in a parasite strain overexpressing recombinant TgATG6 fused with mCherry.By using CRISPR/Cas9 method,we generated TgATG6-deleted parasites were generated.The effect on development of parasites caused by TgATG6 deficiency was analysed by flow cytometry.The results suggested that TgATG6 had close relationship with plant ATG6 and TgATG6 signal was found in the cytosol of intracellular parasites and accumulated into spots in starvation-treated parasites.Sequencing analysis indicated that two TgATG6-deficient strains were obtained in this study(△TgATG6-1 and ATgATG6-2).In ATgATG6-2 strain,a 226 bp sequence was integrated into the target site (593 bp-612 bp),whereas the target sequence could not be amplified in ATgATG6-1 strain.These results demonstrated that TgATG6 was deleted by insertion inactivation in both of the strains.Deletion of TgATG6 caused a defect in invasion ability of T.gondii.Construction of the TgATG6 knockout strain laid a foundation for further investigation of its functions.%酵母ATG6蛋白是磷脂酰基醇-3激酶(PI3K)复合物Ⅰ的组成成分,在自噬起始阶段促进自噬小体的形成.为研究弓形虫ATG6同源蛋白(TgATG6)生物学功能,本研究首先构建进化树对TgATG6基因进行同源性分析,并通过构建稳定表达红色荧光的(ATG6-mCherry)虫株,以检测TgATG6的亚细胞定位情况.通过CRISPR/Cas9技术制备TgATG6基因缺失虫株,采用流式细胞术对该虫株的生长特性进行检测.实验结果显示,TgATG6基因在进化关系上与植物来源的ATG6同源关系较近.TgATG6在虫体内呈弥散分布,经16h饥饿诱导后,该蛋白在弓形虫体内发生聚集.经序列分析显示,成功获得了两种TgATG6基因缺失虫株(△TgATG6-1和△TgATG6-2).在△TgATG6-2虫株中,TgATG6基因开放阅读框的593～612位点之间插入了226 bp外源序列,而在△TgATG6-2中,不能够有效扩增靶序列,表明两种突变虫株的TgATG6基因均被有效插入失活.此外,该基因的缺失可能影响了虫株侵入宿主细胞的能力.TgATG6基因缺失株的成功制备为进一步研究TgATG6蛋白功能奠定了基础.
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