METHODS: The full-length cDNA, gDNA and promoter region of Ss-nobp-1 was identified using protein BLAST in WormBase ParaSite according to the Caenorhabditis elegans NOBP-1 sequence to analyze the gene structure. RNA sequencing (RNA-seq) data in wormbase were retrieved and analyzed to assess the transcript abundance of Ss-nobp-1 in seven developmental stages of S. stercoralis. The standard method for gonadal microinjection of constructs was carried out to determine the anatomic expression patterns of Ss-nobp-1. The interaction between Ss-NOBP-1 and partner of NOBP-1 (Ss-PNO-1) was assessed by yeast two-hybridization and bimolecular fluorescence complementarity (BiFC) experiments.
RESULTS: The NOBP-1 encoding gene Ss-nopb-1 from the zoonotic parasite S. stercoralis has been isolated and characterized. The genomic DNA representing Ss-nobp-1 includes a 1599-bp coding region and encodes a protein comprising 403 amino acids (aa), which contains conserved PIN domain and zinc ribbon domain. RNA-seq analysis revealed that Ss-nobp-1 transcripts are present throughout the seven developmental stages in S. stercoralis and have higher transcription levels in iL3, L3 and P Female. Ss-nobp-1 is expressed mainly in the intestine of transgenic S. stercoralis larvae, and there is a direct interaction between Ss-NOBP-1 and Ss-PNO-1.
CONCLUSIONS: Collectively, Ss-NOBP-1 has a potential role in embryo formation and the infective process, and findings from this study provide a sound foundation for investigating its function during the development of parasitic nematode.
方法:全长cDNA,根据秀丽隐杆线虫NOBP-1序列,使用WormBaseParaSite中的BLAST蛋白鉴定了Ss-nobbp-1的gDNA和启动子区,以分析基因结构。检索并分析了蠕虫中的RNA测序(RNA-seq)数据,以评估S.stercoralis的七个发育阶段中Ss-nobp-1的转录本丰度。进行构建体的性腺显微注射的标准方法以确定Ss-nobp-1的解剖表达模式。通过酵母双杂交和双分子荧光互补(BiFC)实验评估了Ss-NOBP-1与NOBP-1伴侣(Ss-PNO-1)之间的相互作用。
结果:已分离并鉴定了人畜共患寄生虫S.stercoralis的NOBP-1编码基因Ss-nopb-1。代表Ss-nobbp-1的基因组DNA包括1599bp的编码区,并编码包含403个氨基酸(aa)的蛋白质,其中包含保守的PIN域和锌带域。RNA-seq分析显示,Ss-nobbp-1转录本存在于子宫链球菌的七个发育阶段,并且在iL3,L3和P雌性中具有更高的转录水平。Ss-nobbp-1主要在转基因胸骨链球菌幼虫的肠道中表达,Ss-NOBP-1和Ss-PNO-1之间存在直接相互作用。
结论:总的来说,Ss-NOBP-1在胚胎形成和感染过程中具有潜在的作用,这项研究的发现为研究其在寄生线虫发育过程中的功能提供了坚实的基础。