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目的研究日本血吸虫新基因Sj79的结构特征及特点,并进行RNA干扰以观察其RNAi效应,为进一步研究其在抗血吸虫生殖发育中的作用及机制提供依据。方法利用NCBI数据库中已公开的日本血吸虫EST序列信息对Sj79的分子结构特征进行分析,并对不同虫期和性别血吸虫的Sj79的mRNA表达水平进行分析,对雌虫进行Sj79的siRNA浸泡干扰,干扰后采用定量PCR检测Sj79转录本相对表达水平变化。结果 Sj79蛋白的开放阅读框含有696对碱基,由一个外显子构成。该基因具有明显的期特异性,在雌虫中转录水平较高。siRNA片段浸泡3 d后,低剂量(20 nmol/L)siRNA-1组Sj79转录本的表达水平[(41.0±12.3)%]低于siRNA-NC组[(103.2±14.4)%],差异有统计学意义(t=3.28,P<0.05);高剂量(200 nmol/L)siRNA-1组[(15.8±10.9)%]、siRNA-2组[(11.1±8.8)%]虫体Sj79转录本的相对表达水平均低于siRNA-NC组[(100.1±6.3)%],差异均有统计学意义(t=13.44、27.84,P均<0.01),前二者对Sj79转录本的转录抑制率可达84.2%、88.9%。si RNA片段浸泡7 d后,仅低剂量siRNA-1[(43.4±4.5)%]、siRNA-2[(62.5±5.4)%]干扰后虫体Sj79转录本的相对表达水平低于siRNA-NC组[(100.4±5.2)%](t=8.33、5.07,P均<0.01)。结论本研究完善了Sj79基因的mRNA序列和基因组信息,了解了其结构特征,并用siRNA干扰法筛选到了有效的si RNA-1和si RNA-2片段,有利于后续观察Sj79分子干扰对体外雌虫产卵的影响。
Objective To study the structural characteristics and characteristics of Sj79, a novel gene of Schistosoma japonicum, and to observe its RNAi effect by RNA interference, so as to provide evidence for further study on its role and mechanism in anti-schistosoma reproductive development. Methods The molecular structure characteristics of Sj79 were analyzed by using published EST sequences of Schistosoma japonicum in the NCBI database. The mRNA expression levels of Sj79 in different stages and genders of schistosomiasis were analyzed. Sj79 siRNA interference and interference Quantitative PCR was used to detect the relative expression level of Sj79 transcript. Results The open reading frame of Sj79 protein contains 696 pairs of bases and consists of one exon. The gene has obvious period specificity, higher transcription level in females. The mRNA level of Sj79 in low dose (20 nmol / L) siRNA-1 group was significantly lower than that in siRNA-NC group [(41.0 ± 12.3)%] [(103.2 ± 14.4)%] (15.8 ± 10.9%) in siRNA-1 group and [(11.1 ± 8.8)%] in siRNA-2 group were significantly higher than those in control group (t = 3.28, P < The relative expression level of this gene was lower than that of the siRNA-NC group [(100.1 ± 6.3)%], the differences were statistically significant (t = 13.44, 27.84, P <0.01) Rate up to 84.2%, 88.9%. The relative expression level of Sj79 transcript in the RNAi-only siRNA group was lower than that of the siRNA-2 siRNA-2 siRNA group after 7 days of soaking with si RNA fragment ([43.4 ± 4.5]%] and siRNA-2 [(62.5 ± 5.4) Group [(100.4 ± 5.2)%] (t = 8.33,5.07, P <0.01). Conclusions This study improved the mRNA and genomic information of Sj79 gene, understood its structural characteristics, and screened the effective si RNA-1 and si RNA-2 fragments by siRNA interference, which is conducive to follow-up observation of Sj79 molecular interference on in vitro females The effects of spawning.