日本血吸虫平衡型核苷转运蛋白3基因(SjENT3)的RNA干扰研究

Study on RNA Interference of Equilibrative Nucleoside Transporter 3 Gene(SjENT3)of Schistosoma japonicum

血吸虫以雌雄合抱的方式生存,雌虫在合抱后才能发育成熟,雄虫通过某种非精子传递的机制调控血吸虫雌虫的生长发育,而成熟雌虫所产大量虫卵是血吸虫导致宿主严重病理损害和疾病再传播的根源,因此,了解雄虫对雌虫发育的调控对血吸虫的致病和传播具有重要意义.本实验室前期蛋白质组学研究结果显示日本血吸虫ENT3蛋白(SjENT3)在两性感染的合抱雄虫体内的表达高于其在单性感染雄虫体内的表达.本研究通过qRT-PCR发现SjENT3基因在日本血吸虫虫体各个检测时间点均转录,从7d到21d逐渐升高,在21d转录水平达到最高,随后逐渐下降;SjENT3基因在18,21,25,28,35,42d在合抱雄虫体内转录水平明显高于合抱雌虫;对合抱雄虫与单性感染雄虫转录水平分析发现其在21d,23d,25d 3个检测时间点与蛋白质组学结果一致,在合抱雄虫体内高表达.利用筛选出效果较好的siRNA对SjENT3在血吸虫体内的转录进行长期干扰,结果发现:特异siRNA干扰显著影响SjENT3转录水平,和NC对照组相比,干扰引起宿主虫荷率下降24.44%(P<0.05),宿主肝脏荷卵率下降57.47%(P<0.01),干扰后雌虫肝产卵能力下降42.28%(P<0.01).本研究结果初步表明SjENT3基因参与日本血吸虫尤其是雄虫的生长发育,并可能与血吸虫雌雄虫合抱及其相互间信息传递有一定的关系.

Schistosomes are gonochorous,the mating of male and female worms can stimulate the female sexual mature,and these stimuli are independent of sperm transfer.The large amount of eggs produced by mature female worms not only induce major pathological damage to hosts of schistosomes but also lead to disease transmission.Therefore,it is important to understand the regulation of male on female of schistosomes.Our previous study showed that the expression of SjENT3 protein in bisexual infected males are higher than in single-sex infected males.In this study,it was found through qRT-PCR that the SjENT3 gene was transcribed at every detection time point of Schistosoma japonicum,it was gradually increased from day 7 to day 21,reaching the highest transcription level at day 21,and then decreased gradually;The transcription of SjENT3 are higher in mated males than mated females at day 18,21,25,28,35 and 42 after infection;its transcription level are higher in bisexual infected males than in single-sex infected males at day 21,23,25 after infection,which is consistent with previous proteomics results.The functions of SjENT3 were explored via a small interfering RNA-based gene silencing approach.The results showed that the long-term interference can down regulated the transcription of SjENT3.RNA interference also affected parasitic vitality,development,and reproduction.Compared with the NC groups,RNA interference caused a 24.44%reduction in the host's worm load,the reproductive capacity of female decreased 42.28%,then induced 57.47%reduction of the liver egg load in host.The results indicated that the SjENT3 may involve in the growth and development of Schistosoma japonicum,especially the males,and may have a certain relationship with the mating and the information transmission between female and male worms.