Prophenoloxidase (proPO) is very important to protect the invertebrates from microbial infections. Our previous studies revealed that proPO was up‐regulated in WSSV‐injected Macrobrachium rosenbergii and is responsible for protecting M. rosenbergii from WSSV. In order to prove this mechanism, an attempt was made in the present study to silence the proPO gene in freshwater prawn by injection of dsRNA‐proPO followed by WSSV challenge. Two partial fragments of proPO with the size of 251 and 331 bp were used to synthesize dsRNA using LITMUS38i vector and E. coli. The bacterially synthesized dsRNA‐proPO was used to silence proPO gene to determine its involvement in developing resistance in prawn against WSSV. In proPO gene‐silenced prawn, 100% mortality was observed after WSSV challenge whereas no mortality was observed in prawn injected with WSSV alone. The WSSV infection in gene‐silenced prawn was confirmed by PCR, and its propagation was quantified by ELISA and real‐time PCR at different time intervals. Real‐time PCR assay revealed a significant reduction in the expression of proPO gene in WSSV‐challenged proPO‐silenced prawn when compared to normal prawn. Level of proPO was reduced significantly in the haemolymph of proPO‐silenced prawn when compared to prawn injected with PBS.

中文翻译：

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淡水虾罗氏沼虾中的苯酚氧化酶（proPO）基因沉默，使它们容易感染白斑综合症病毒（WSSV）

酚氧化酶（proPO）对于保护无脊椎动物免受微生物感染非常重要。我们以前的研究表明，proPO在WSSV注射的罗氏沼虾中被上调，并负责保护罗氏沼虾免受WSSV侵害。为了证明这种机制，本研究尝试通过注入dsRNA-proPO，然后进行WSSV攻击来沉默淡水虾中的proPO基因。使用LITMUS38i载体和大肠杆菌，使用251和331 bp大小的proPO的两个部分片段合成dsRNA 。。细菌合成的dsRNA-proPO用于沉默proPO基因，以确定其参与虾对WSSV的抗性发展。在用proPO基因沉默的虾中，WSSV攻击后观察到100％的死亡率，而单独注射WSSV的虾则未观察到死亡率。通过PCR证实了WSSV在基因沉默大虾中的感染，并通过ELISA和实时PCR在不同的时间间隔对WSSV的传播进行了定量。实时PCR分析显示，与普通虾相比，WSSV挑战的proPO沉默的虾中proPO基因的表达显着降低。与注射PBS的对虾相比，proPO沉默的对虾的血淋巴中proPO的水平显着降低。