日本血吸虫Sj79基因的分子特征及RNA干扰效率

[Molecular characteristics and RNA interference efficiency of Schistosoma japonicum Sj79 gene].

作者信息

Jiang Yan-yan, Yuan Zhong-ying, Xu Yu-xin, Zang Wei, Cao Jian-ping, Wang Ying, Yin Jian-hai, Shen Yu-juan

出版信息

Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi. 2015 Jun;27(3):273-6, 289.

Abstract

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 developmental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The transcriptional 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-1 group [(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 significance (t = 8.33, 5.07, both P < 0.01).

CONCLUSION

Through this study, we have improved the mRNA sequence and genomic information of Sj79 gene, and understood its structural features, as well as selected out two effect fragments siRNA-1 and siRNA-2, which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schistosome in vitro.

摘要

目的

研究日本血吸虫新基因Sj79的结构特征，观察其RNA干扰（RNAi）效果，为进一步研究其功能及血吸虫抗生殖发育机制提供实验依据。

方法

采用生物信息学方法分析Sj79基因结构及特征。分析Sj79信使核糖核酸（mRNA）在血吸虫不同发育阶段的表达情况，并采用浸泡法观察RNAi沉默效果。通过实时荧光定量PCR检测RNAi后Sj79的转录水平。

结果

Sj79的开放阅读框含696个碱基对，具有外显子结构。该基因具有明显的阶段特异性，在成熟雌虫中转录水平最高。浸泡3 d后，低剂量（20 nmol/L）的siRNA-1组Sj79 mRNA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）时，siRNA-1组[（15.8±10.9）%]和siRNA-2组[（11.1±8.8）%]的Sj79 mRNA水平均显著低于siRNA-NC组[（100.1±6.3）%]（t = 13.44、27.84，P均< 0.01）。浸泡7 d后，仅低剂量的siRNA-1组[（43.4±4.5）%]和siRNA-2组[（62.5±5.4）%]的Sj79 mRNA水平低于siRNA-NC组[（100.4±5.2）%]，差异有统计学意义（t = 8.33、5.07，P均< 0.01）。