Silva Marta G, Knowles Donald P, Suarez Carlos E
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA.
Animal Disease Research Unit, Agricultural Research Service, USDA, WSU, Pullman, WA, USA.
Parasit Vectors. 2016 Nov 11;9(1):576. doi: 10.1186/s13071-016-1859-9.
Tick-borne Babesia bigemina is responsible for acute and potentially lethal hemolytic disease in cattle. The development of genetic manipulation tools necessary to the better understanding of parasite biology is currently limited by the lack of a complete parasite genome and experimental tools such as transfection. Effective promoters, required to regulate expression of transgenes, such as the elongation factor-1 alpha (ef-1α), have been identified in other apicomplexans such as Babesia bovis and Plasmodium falciparum.
The B. bigemina ef-1a locus was defined by searching a partial genome library of B. bigemina (Sanger Institute). Presence of an intron in the 5' untranslated region was determined by 5' Rapid Amplification of cDNA Ends (RACE) analysis. Promoter activity was determined by measurement of luciferase expression at several time points after electroporation, efficiency of transfections and normalization of data was determined by quantitative PCR and by the percentage of parasitized erythrocytes.
The ef-1α locus contains two identical head to head ef-1α genes separated by a 1.425 kb intergenic (IG) region. Significant sequence divergence in the regions upstream of the inverted repeats on each side of the B. bigemina IG region suggest independent regulation mechanisms for controlling expression of each of the two ef-1α genes. Plasmid constructs containing the 5' and 3' halves of the IG regions controlling the expression of the luciferase gene containing a 3' region of a B. bigemina rap-1a gene, were generated for the testing of luciferase activity in transiently transfected parasites. Both halves of the ef-1α IG region tested showed the ability to promote high level production of luciferase. Moreover, both B. bigemina ef-1α promoters are also active in transiently transfected B. bovis and conversely, a B. bovis ef-1α promoter is active in transiently transfected B. bigemina.
Collectively these data demonstrate the existence of two distinct promoters with homologous and heterologous promoter function in B. bigemina and B. bovis which is described for the first time in Babesia species. This study is of significance for development of interspecies stable transfection systems for B. bigemina and for B. bovis.
蜱传播的双芽巴贝斯虫可导致牛的急性且可能致命的溶血性疾病。目前，由于缺乏完整的寄生虫基因组以及诸如转染等实验工具，限制了深入了解寄生虫生物学所需的基因操作工具的开发。在其他顶复门寄生虫如牛巴贝斯虫和恶性疟原虫中已鉴定出调控转基因表达所需的有效启动子，如延伸因子-1α（ef-1α）。
通过搜索双芽巴贝斯虫的部分基因组文库（桑格研究所）来确定双芽巴贝斯虫ef-1a基因座。通过5' cDNA末端快速扩增（RACE）分析确定在5'非翻译区是否存在内含子。在电穿孔后的几个时间点通过测量荧光素酶表达来确定启动子活性，通过定量PCR以及被寄生红细胞的百分比来确定转染效率和数据标准化。
ef-1α基因座包含两个相同的头对头ef-1α基因，由一个1.425 kb的基因间隔（IG）区域隔开。双芽巴贝斯虫IG区域两侧反向重复序列上游区域存在显著的序列差异，提示这两个ef-1α基因的表达调控机制相互独立。构建了包含控制荧光素酶基因表达的IG区域5'和3'部分的质粒构建体，该荧光素酶基因含有双芽巴贝斯虫rap-1a基因的3'区域，用于检测瞬时转染寄生虫中的荧光素酶活性。所测试的ef-1α IG区域的两个部分均显示出促进荧光素酶高水平产生的能力。此外，双芽巴贝斯虫的两个ef-1α启动子在瞬时转染的牛巴贝斯虫中也具有活性，反之，牛巴贝斯虫的一个ef-1α启动子在瞬时转染的双芽巴贝斯虫中也具有活性。
总体而言，这些数据证明在双芽巴贝斯虫和牛巴贝斯虫中存在两个具有同源和异源启动子功能的不同启动子，这在巴贝斯虫属中尚属首次描述。本研究对于双芽巴贝斯虫和牛巴贝斯虫种间稳定转染系统的开发具有重要意义。
