日本血吸虫新基因Sj79的分子特征及RNA干扰效应  

Molecular characteristics and RNA interference efficiency of Schistosoma japonicum Sj79 gene

作　　者：姜岩岩[1] 袁忠英[1] 徐馀信[1] 臧炜[1] 曹建平[1] 王莹[1] 尹建海[1] 沈玉娟[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海200025

出　　处：《中国血吸虫病防治杂志》2015年第3期273-276,289,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(30771880;81371841;81371842)

摘　　要：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 features and characteristics of a novel gene Schistosoma japonicum 79（Sj79）, and observe its effect of RNA interference（RNAi）,so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. Methods The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA（mRNA）during the different develop-mental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The tran-scriptional levels of Sj79 after RNAi were detected by real time PCR. Results The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity,and its transcriptional level in mature female worms was the highest. After soaking for 3 d,the Sj79 mRNA level[（41.0 ± 12.3）%]in the siRNA-1 group with low dosage（20 nmol/L） was lower than that in the siRNA-NC group[（103.2 ± 14.4）%],the difference was statistically significant（t=3.28,P〈0.05）. When with high dosage（200 nmol/L ）,both the Sj79 mRNA levels in the siRNA-1 group[（15.8 ± 10.9）%]and siRNA-2 group [（11.1 ± 8.8）%]were significantly lower than that in the siRNA-NC group[（100.1 ± 6.3）%]（t=13.44,27.84,both P〈0.01）. After soaking for 7 d,only the Sj79 mRNA levels in the siRNA-1group[（43.4 ± 4.5）%]and siRNA-2 group[（62.5 ± 5.4）%]with low dosage were lower than that in the siRNA-NC group[（100.4 ± 5.2）%],and the differences had statistical sig-nificance（t=8.33,5.07,both P〈0.01）. Conclusion Through this study,we have improved the mRNA sequence and genom-ic information of Sj79 gene,and understood its structural features,as well as selected out two effect fragments siRNA-1and siR- NA-2 which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schisto-some in vitro.

关 键 词：日本血吸虫 Sj79 分子特征 RNA干扰 

分 类 号：R383.24[医药卫生—医学寄生虫学]